Drug development target protein and target gene, and method of screening

ABSTRACT

The present invention provides novel target proteins and target genes for drug discovery, and the means that enable the development of novel drugs using the same. More particularly, the present invention provides NCS proteins and genes thereof; screening methods for drug (for example, anti-central nervous disease drug); agents for regulating disease (for example, central nervous disease); production methods of a drug derivative; a complex comprising a drug and NCS protein, and a method of producing the complex; a kits comprising a drug or a salt thereof; determination methods for the onset or risk of onset of a specified disease, determination methods for susceptibility to a drug, and determination kits used for the determination methods; and the like.

INCORPORATION-BY-REFERENCE OF MATERIAL ELECTRONICALLY SUBMITTED

Incorporated by reference in its entirety herein is a computer-readablenucleotide/amino acid sequence listing submitted concurrently herewithand identified as follows: 11,078 bytes ASCII (Text) file named“701552SequenceListing.txt,”created Apr. 18,2007.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to target proteins and target genes thatare useful for the development of drugs such as anti-central nervousdisease drug; a screening method for drugs such as anti-central nervousdisease drug, and the substance obtained by the screening method; anagent of regulating a pharmacological action such as central nervousaction; a drug derivative and a method of producing the derivative; anda complex comprising a drug and a target protein thereof, and a methodof producing the complex, and the like.

BACKGROUND ART

Alzheimer's disease (AD) is a type of dementia that affects more than 15million people of the entire world, and is predicted to affect more inthe future, as the average lifetime increases. On the other hand, Downsyndrome is a hereditary disease which occurs because of chromosome 21trisomy (3 copies), and it is known that as Down syndrome patients grow,various AD-like changes in the brain gradually show up, and many Downsyndrome patients in their middle ages are affected by AD. Preventingdementia is an important challenge in the aging society, and aneffective preventive is strongly desired.

Statins, which are HMG-CoA (3-hydroxy-3-methylglutarylcoenzyme A)reductase inhibitor cholesterol-lowering agents, have been shown byepidemiological studies to have the effect of greatly lowering theoccurrence rate of Alzheimer's disease, and expectations for dementiaprevention by statins are rising. However, the action mechanism of LDLcholesterol decrease and AD are still unknown, rather a target moleculeother than HMG-CoA reductase is speculated to be involved in the declineof AD occurrence rate, and it is necessary to clarify thedementia-related target molecule(s) of statins in order to carry outlogical drug discovery for dementia prevention.

In recent years, on the other, the genome sequences of a variety oforganisms have been elucidated and analyzed at the global level. For thehuman genome, in particular, a worldwide cooperative research projectwas implemented, and completion of analysis of all sequences thereof wasannounced in April 2003. As a result, it is becoming possible to analyzecomplex biological phenomena in the context of the functions and controlof all genes, or networks of gene-gene, protein-protein, cell-cell, andindividual-individual interactions. The genome information thus obtainedhas been significantly revolutionizing a number of industries, includingdrug development, as well as in academic sectors.

For example, it has been reported that there are about 480 kinds oftarget proteins for drugs having been in common use to date, and thatthese target proteins are limited to membrane receptors, enzymes, ionchannels, or nuclear receptors and the like (J. Drews, Science, 287,1960-1964, 2000). Meanwhile, target protein search based on genomeinformation has discovered an extremely large number of target proteins,including novel proteins not covered in the conventional range of targetproteins one after another, which are estimated to total about 1,500kinds (A. L. Hopkins & C. R. Groom, Nature Reviews; Drug Discovery, 1,727-730, 2002).

However, despite the fact that the research and development expendituresspent by pharmaceutical companies are increasing due to rises ininfrastructuring costs for coping with vast amounts of data like genomeinformation and clinical developmental costs, the number of new drugsapproved per year is tending to decrease on the contrary (NatureReviews; Drug Discovery, February, 2003). This shows that theabove-described genome information is actually not efficiently utilized.

As a means for overcoming these circumstances, Nagashima et al. invented“Method, System, Apparatus, and Device for Discovering and PreparingChemical for Medical and Other Uses” and filed a patent application forthat invention (Japanese Patent Kohyo Publication No. 2004-509406).

Disclosed in that patent application are methods, systems, databases,user interfaces, software, media, and services that are useful for theevaluation of compound-protein interactions, and are also useful for theutilization of the information resulting from such an evaluationintended to discover compounds in medical and other areas. Furthermore,it is intended to produce a very large pool of novel target proteins fordrug discovery, novel methods for designing novel drugs, and a pool ofsmall substances for therapeutic purposes that are virtually synthesizedas having been inconceivable in the past.

Specifically, disclosed in that patent application were a method ofidentifying a protein or partial protein that is appropriate as a noveldrug discovery target, which comprises the following steps:

-   (i) a step for selecting a plurality of proteins or partial proteins    showing desired affinity and specificity for a selected target    compound;-   (ii) a step for identifying the structure and function of the    protein or the partial protein; and-   (iii) a step for selecting a single protein or single partial    protein having a desired function, and a method of discovering a    drug, which comprises the following steps:-   (i) a step for investigating the chemical structure of the target    compound selected using the above-described method; and-   (ii) a step for chemically modifying the structure of the selected    target compound to optimize the affinity and specificity of the    modified compound for the protein or the partial protein, which is    appropriate as a novel drug target.

Furthermore, another feature of the method disclosed in that patentapplication resides in that the selected target compound is a compoundapproved for medical use.

Conventional drugs that have been used to date include many drugs forwhich target proteins are unknown, or for which target proteins areknown but not all of whose pharmacological effects and adverse effectscan be explained by mechanisms mediated by the proteins.

Typically, aspirin, one of the drugs that have longest been used, may bementioned. When aspirin was launched in the market for the first timemore than 100 years ago, the mechanism for its anti-inflammatory actionwas unclear. About 70 years later, aspirin was found to havecyclooxygenase (COX) inhibitory action. Still 20 years later, it wasdemonstrated that COX occurred in two subtypes: COX-1 and COX-2, thatthe primary pharmacological effect of aspirin was based on COX-2inhibition, and that COX-1 inhibitory action was the cause of adverseeffects such as gastrointestinal disorders. However, not all the targetproteins for aspirin have been elucidated. In recent years, aspirin hasbeen shown to exhibit anticancer action and antidementic action inclinical settings, but these pharmacological effects cannot be explainedby COX inhibition. On the other, recent years have seen many papersreporting that aspirin acts on transcription factors such as IKKβ and onnuclear receptors such as PPAR-γ, but the relationship between these andthe various pharmacological effects of aspirin remains unclear.

For these reasons, elucidating target proteins for traditionally useddrugs can be said to be a very effective approach to discovering noveldrug discovery target proteins.

Hirayama, one of the inventors of the above-described published patent,and others generated a database integrating the structural and physicalproperty data on about 1,500 kinds of drugs commercially available inJapan, and found that existing pharmaceutical compounds share structuralfeatures (Chem-Bio Informatics Journal, 1, 18-22, 2001). Drugs that havebeen commonly used to date can be described as excellent in that theyhave cleared the issues of localization in the body and safety in theirdevelopmental processes. Searching novel target proteins with theseexisting drugs as probes, and selecting novel new drug candidatecompounds on the basis of their structures is thought to be a highlyreasonable and efficient approach.

A second problem arises concerning how to make use of the genomeinformation during the search for novel target proteins. Solelydetermining the genome sequence is not sufficient to ensure theelucidation of the functions of all genes and the discovery of drugdiscovery target proteins. It is estimated that in humans, about 30,000to 40,000 kinds of genes are present; taking into consideration variantsfrom alternative splicing, there are reportedly more than 100,000 kindsof mRNA. It is important, therefore, that out of the vast amount of newgenes revealed from the genome sequence, those having useful functionsin industrial applications, including drug development, should beefficiently selected and identified.

In many cases of the genome sequences of eukaryotic organisms, each geneis divided into a plurality of exons by introns; therefore, it isimpossible to accurately predict the structure of the protein encoded bythe gene solely from the sequence information on the gene. In contrast,for a cDNA prepared from intron-excluded mRNA, information on the aminoacid sequence of protein is obtained as information on a singlecontinuous sequence, enabling easy determination of the primarystructure thereof.

In particular, analyzing a full-length cDNA enables the identificationof the mRNA transcription initiation point on the genome sequence basedon the 5′-terminal sequence of the cDNA, and also enables analysis offactors involved in the stability of mRNA contained in the sequence andthe expression control in the translation stage. Also, because the ATGcodon, which serves as the translation initiation point, is present onthe 5′ side, translation into protein in the right frame can beachieved. Therefore, by using an appropriate gene expression system, itis also possible to mass-produce the protein encoded by the cDNA, and toexpress the protein and analyze the biological activity thereof. Hence,it is considered that by performing an analysis using a proteinexpressed from full-length cDNA, important information that could not beobtained solely by genome sequence analysis is obtained, and that it ispossible to discover novel target proteins that do not lie in theconventional category of drug discovery target proteins.

USP publication no. 20030159158 (Nef, Patrick, Aug. 21, 2003) disclosesa screening method for an NCS1 agonist targeting NCS1, which is a kindof NCS. However, there is a plurality of molecular species of NCS, whichis expressed specifically or complementarily in various tissues such asthe cerebral nerve tissue, secretory tissue, immuno-related cells,epithelium of blood vessels, and relates to many functions. Hence,screening or designing directed to compounds with structures that arepreferred for NCS binding is necessary in order to efficiently producenew pharmaceutical compounds that target the NCS family. The presentinvention discloses preferred structures for NCS binding, of which manyin addition have structural motifs that exist in common drugs. For thisreason, by taking the structure disclosed in the present invention asthe starting point, it will be possible to efficiently screen or designa compound of high drug efficacy and safety. Furthermore, a screeningmethod targeting NCS1 is disclosed in USP publication 20030159158.However, the NCS-binding compound and its screening method in thepresent application is directed to neurocalcin δ that belongs to theVILIPS family (Class B) and human hippocalcin-like protein 1 (orVisinin-like protein 3 or VILIP-3), which are indicated to be related tocentral nervous diseases, especially to dementia such as Alzheimer'sdisease, making it especially preferable for the development oftherapeutic drugs for central nervous diseases centered around dementia.Note however, that the NCS-binding compound disclosed in the presentinvention is not limited to the VILIPS family (Class B), but comprisescompounds that bind to the whole NCS family, including NCS 1.

DISCLOSURE OF THE INVENTION

The objects of the present invention are to provide target proteins fordrug discovery and compounds binding thereto, target genes for drugdiscovery, the various means that enable the development of novelmedicines utilizing the above, and the like.

The present inventors diligently investigated new drug discovery targetproteins that can be useful for the development of new drugs, byanalyzing by the SEC/MS method, the interactions between human proteinsand compounds that have been used as drugs, and found that theneuron-specific calcium ion sensor protein (NCS protein) can be a targetprotein for creating a drug such as an anti-central nervous diseasedrug. Based on these findings, the present inventors conceived thatcompounds capable of binding to the NCS protein, and substances whichregulate the expression or function of the NCS protein gene aresubstances which can be useful as drugs, and that in order to developdrugs such as anti-central nervous disease drugs, we can screensubstances capable of binding to the NCS protein, or substances thatregulate the expression or function of the NCS protein gene, or wederivatize drugs so as to be able to regulate the function of the NCSprotein by binding thereto, or to regulate the expression or function ofthe NCS protein gene, and completed the present invention.

Accordingly, the present invention is as follows:

-   [1] A compound capable of binding to an NCS protein, which is    selected from the group consisting of the following formulas (I) to    (VIII), or a salt thereof;

wherein

-   R¹ is a hydrogen atom; a halogen atom; cyano; hydroxy; straight    chain or branched alkyl having 1 to 7 carbon atoms; halogenated    alkyl; alkyloxy; alkylsulfanyl; straight chain or branched alkyl    having 1 to 5 carbon atoms, straight chain or branched alkyloxy    having 1 to 5 carbon atoms, phenyl, phenylsulfanyl, phenylalkyl    having 7 to 12 carbon atoms, phenylalkenyl having 8 to 12 carbon    atoms or phenylalkyloxy having 7 to 12 carbon atoms, each of which    optionally has 1 to 3 substituents selected from the group    consisting of a halogen atom, cyano, hydroxy, amino,    mono-substituted amino, di-substituted amino, straight chain or    branched alkyl having 1 to 5 carbon atoms, halogenated alkyl,    alkyloxy and alkylsulfanyl; or —CO—R⁹ (wherein R⁹ is straight chain    or branched alkyl having 1 to 9 carbon atoms; or phenyl, cycloalkyl    having 3 to 7 carbon atoms, phenylalkyl having 7 to 11 carbon atoms,    imidazolyl, biphenyl, thienyl, benzothienyl or benzofuryl, each of    which optionally has 1 to 3 substituents selected from the group    consisting of straight chain or branched alkyl having 1 to 9 carbon    atoms, a halogen atom, cyano, hydroxy, amino, mono-substituted    amino, di-substituted amino, alkylsulfanyl, halogenated methyl and    4-hydroxyphenyl),-   R² is a hydrogen atom; a halogen atom; cyano; hydroxy; straight    chain or branched alkyl having 1 to 7 carbon atoms; halogenated    alkyl; alkyloxy; alkylsulfanyl; or straight chain or branched alkyl    having 1 to 5 carbon atoms, straight chain or branched alkyloxy    having 1 to 5 carbon atoms, phenyl, phenyloxy, phenylsulfanyl,    phenylalkyl having 7 to 12 carbon atoms, phenylalkenyl having 8 to    12 carbon atoms or phenylalkyloxy having 7 to 12 carbon atoms, each    of which optionally has 1 to 3 substituents selected from the group    consisting of a halogen atom, cyano, hydroxy, amino,    mono-substituted amino, di-substituted amino, straight chain or    branched alkyl having 1 to 5 carbon atoms, halogenated alkyl,    alkyloxy and alkylsulfanyl,-   R³ is a hydrogen atom; a halogen atom; cyano; hydroxy; straight    chain or branched alkyl having 1 to 7 carbon atoms; halogenated    alkyl; alkyloxy; alkylsulfanyl; or straight chain or branched alkyl    having 1 to 5 carbon atoms, straight chain or branched alkyloxy    having 1 to 5 carbon atoms, phenyl, phenylsulfanyl, phenylalkyl    having 7 to 12 carbon atoms, phenylalkenyl having 8 to 12 carbon    atoms or phenylalkyloxy having 7 to 12 carbon atoms, each of which    optionally has 1 to 3 substituents selected from the group    consisting of a halogen atom, cyano, hydroxy, amino,    mono-substituted amino, di-substituted amino, straight chain or    branched alkyl having 1 to 5 carbon atoms, halogenated alkyl,    alkyloxy and alkylsulfanyl,-   R⁴ is a hydrogen atom; a halogen atom; cyano; hydroxy; straight    chain or branched alkyl having 1 to 7 carbon atoms; halogenated    alkyl; alkyloxy; alkylsulfanyl; or straight chain or branched alkyl    having 1 to 5 carbon atoms, straight chain or branched alkyloxy    having 1 to 5 carbon atoms, phenyl, phenylsulfanyl, phenylimino,    phenylalkyl having 7 to 12 carbon atoms, phenylalkenyl having 8 to    12 carbon atoms or phenylalkyloxy having 7 to 12 carbon atoms, each    of which optionally has 1 to 3 substituents selected from the group    consisting of a halogen atom, cyano, hydroxy, amino,    mono-substituted amino, di-substituted amino, straight chain or    branched alkyl having 1 to 5 carbon atoms, halogenated alkyl,    alkyloxy and alkylsulfanyl,-   R⁵ is a hydrogen atom; a halogen atom; cyano; straight chain or    branched alkyl having 1 to 5 carbon atoms; or halogenated alkyl,-   R⁶ is a hydrogen atom; a halogen atom; cyano; hydroxy; straight    chain or branched alkyl having 1 to 7 carbon atoms; halogenated    alkyl; alkyloxy; alkylsulfanyl; or straight chain or branched alkyl    having 1 to 5 carbon atoms, straight chain or branched alkyloxy    having 1 to 5 carbon atoms, phenyl, phenylsulfanyl, phenylalkyl    having 7 to 12 carbon atoms, phenylalkenyl having 8 to 12 carbon    atoms or phenylalkyloxy having 7 to 12 carbon atoms, each of which    optionally has 1 to 3 substituents selected from the group    consisting of a halogen atom, cyano, hydroxy, amino,    mono-substituted amino, di-substituted amino, straight chain or    branched alkyl having 1 to 5 carbon atoms, halogenated alkyl,    alkyloxy and alkylsulfanyl,-   R⁷ is a hydrogen atom; a halogen atom; cyano; hydroxy; straight    chain or branched alkyl having 1 to 7 carbon atoms; halogenated    alkyl; alkyloxy; alkylsulfanyl; or straight chain or branched alkyl    having 1 to 5 carbon atoms, straight chain or branched alkyloxy    having 1 to 5 carbon atoms, phenyl, phenylsulfanyl, phenylalkyl    having 7 to 12 carbon atoms, phenylalkenyl having 8 to 12 carbon    atoms or phenylalkyloxy having 7 to 12 carbon atoms, each of which    optionally has 1 to 3 substituents selected from the group    consisting of a halogen atom, cyano, hydroxy, amino,    mono-substituted amino, di-substituted amino, straight chain or    branched alkyl having 1 to 5 carbon atoms, halogenated alkyl,    alkyloxy and alkylsulfanyl, and-   R⁸ is a hydrogen atom; a halogen atom; cyano; hydroxy;    alkylsulfanyl; or straight chain or branched alkyl having 1 to 7    carbon atoms or straight chain or branched alkyloxy having 1 to 7    carbon atoms, each of which optionally has 1 to 3 substituents    selected from the group consisting of a halogen atom, cyano,    hydroxy, halogenated alkyl and alkyloxy,-   provided that each of R² and R⁴ bonded to each other, R³ and R⁶    bonded to each other, R⁶ and R⁷ bonded to each other, and R⁷ and R⁸    bonded to each other may independently forms a ring optionally    having 1 to 3 substituents selected from the group consisting of a    halogen atom; cyano; hydroxy; amino; mono-substituted amino;    di-substituted amino; halogenated alkyl; alkylsulfanyl;    benzimidazolonyl; and straight chain or branched alkyl having 1 to 5    carbon atoms or straight chain or branched alkyloxy having 1 to 5    carbon atoms, each of which optionally has 1 to 3 substituents    selected from the group consisting of a halogen atom, cyano,    hydroxy, amino, mono-substituted amino, di-substituted amino,    alkyloxy and alkylsulfanyl.-   [2] A drug for the treatment or prophylaxis of dementia, which    comprises a compound selected from the group consisting of the    following formulas (I) to (VIII), or a pharmaceutically acceptable    salt thereof, as an active ingredient;

wherein

-   R¹ is a hydrogen atom; a halogen atom; cyano; hydroxy; straight    chain or branched alkyl having 1 to 7 carbon atoms; halogenated    alkyl; alkyloxy; alkylsulfanyl; straight chain or branched alkyl    having 1 to 5 carbon atoms, straight chain or branched alkyloxy    having 1 to 5 carbon atoms, phenyl, phenylsulfanyl, phenylalkyl    having 7 to 12 carbon atoms, phenylalkenyl having 8 to 12 carbon    atoms or phenylalkyloxy having 7 to 12 carbon atoms, each of which    optionally has 1 to 3 substituents selected from the group    consisting of a halogen atom, cyano, hydroxy, amino,    mono-substituted amino, di-substituted amino, straight chain or    branched alkyl having 1 to 5 carbon atoms, halogenated alkyl,    alkyloxy and alkylsulfanyl; or —CO—R⁹ (wherein R⁹ is straight chain    or branched alkyl having 1 to 9 carbon atoms; or phenyl, cycloalkyl    having 3 to 7 carbon atoms, phenylalkyl having 7 to 11 carbon atoms,    imidazolyl, biphenyl, thienyl, benzothienyl or benzofuryl, each of    which optionally has 1 to 3 substituents selected from the group    consisting of straight chain or branched alkyl having 1 to 9 carbon    atoms, a halogen atom, cyano, hydroxy, amino, mono-substituted    amino, di-substituted amino, alkylsulfanyl, halogenated methyl and    4-hydroxyphenyl),-   R² is a hydrogen atom; a halogen atom; cyano; hydroxy; straight    chain or branched alkyl having 1 to 7 carbon atoms; halogenated    alkyl; alkyloxy; alkylsulfanyl; or straight chain or branched alkyl    having 1 to 5 carbon atoms, straight chain or branched alkyloxy    having 1 to 5 carbon atoms, phenyl, phenyloxy, phenylsulfanyl,    phenylalkyl having 7 to 12 carbon atoms, phenylalkenyl having 8 to    12 carbon atoms or phenylalkyloxy having 7 to 12 carbon atoms, each    of which optionally has 1 to 3 substituents selected from the group    consisting of a halogen atom, cyano, hydroxy, amino,    mono-substituted amino, di-substituted amino, straight chain or    branched alkyl having 1 to 5 carbon atoms, halogenated alkyl,    alkyloxy and alkylsulfanyl,-   R³ is a hydrogen atom; a halogen atom; cyano; hydroxy; straight    chain or branched alkyl having 1 to 7 carbon atoms; halogenated    alkyl; alkyloxy; alkylsulfanyl; or straight chain or branched alkyl    having 1 to 5 carbon atoms, straight chain or branched alkyloxy    having 1 to 5 carbon atoms, phenyl, phenylsulfanyl, phenylalkyl    having 7 to 12 carbon atoms, phenylalkenyl having 8 to 12 carbon    atoms or phenylalkyloxy having 7 to 12 carbon atoms, each of which    optionally has 1 to 3 substituents selected from the group    consisting of a halogen atom, cyano, hydroxy, amino,    mono-substituted amino, di-substituted amino, straight chain or    branched alkyl having 1 to 5 carbon atoms, halogenated alkyl,    alkyloxy and alkylsulfanyl,-   R⁴ is a hydrogen atom; a halogen atom; cyano; hydroxy; straight    chain or branched alkyl having 1 to 7 carbon atoms; halogenated    alkyl; alkyloxy; alkylsulfanyl; or straight chain or branched alkyl    having 1 to 5 carbon atoms, straight chain or branched alkyloxy    having 1 to 5 carbon atoms, phenyl, phenylsulfanyl, phenylimino,    phenylalkyl having 7 to 12 carbon atoms, phenylalkenyl having 8 to    12 carbon atoms or phenylalkyloxy having 7 to 12 carbon atoms, each    of which optionally has 1 to 3 substituents selected from the group    consisting of a halogen atom, cyano, hydroxy, amino,    mono-substituted amino, di-substituted amino, straight chain or    branched alkyl having 1 to 5 carbon atoms, halogenated alkyl,    alkyloxy and alkylsulfanyl,-   R⁵ is a hydrogen atom; a halogen atom; cyano; straight chain or    branched alkyl having 1 to 5 carbon atoms; or halogenated alkyl,-   R⁶ is a hydrogen atom; a halogen atom; cyano; hydroxy; straight    chain or branched alkyl having 1 to 7 carbon atoms; halogenated    alkyl; alkyloxy; alkylsulfanyl; or straight chain or branched alkyl    having 1 to 5 carbon atoms, straight chain or branched alkyloxy    having 1 to 5 carbon atoms, phenyl, phenylsulfanyl, phenylalkyl    having 7 to 12 carbon atoms, phenylalkenyl having 8 to 12 carbon    atoms or phenylalkyloxy having 7 to 12 carbon atoms, each of which    optionally has 1 to 3 substituents selected from the group    consisting of a halogen atom, cyano, hydroxy, amino,    mono-substituted amino, di-substituted amino, straight chain or    branched alkyl having 1 to 5 carbon atoms, halogenated alkyl,    alkyloxy and alkylsulfanyl,-   R⁷ is a hydrogen atom; a halogen atom; cyano; hydroxy; straight    chain or branched alkyl having 1 to 7 carbon atoms; halogenated    alkyl; alkyloxy; alkylsulfanyl; or straight chain or branched alkyl    having 1 to 5 carbon atoms, straight chain or branched alkyloxy    having 1 to 5 carbon atoms, phenyl, phenylsulfanyl, phenylalkyl    having 7 to 12 carbon atoms, phenylalkenyl having 8 to 12 carbon    atoms or phenylalkyloxy having 7 to 12 carbon atoms, each of which    optionally has 1 to 3 substituents selected from the group    consisting of a halogen atom, cyano, hydroxy, amino,    mono-substituted amino, di-substituted amino, straight chain or    branched alkyl having 1 to 5 carbon atoms, halogenated alkyl,    alkyloxy and alkylsulfanyl, and-   R⁸ is a hydrogen atom; a halogen atom; cyano; hydroxy;    alkylsulfanyl; or straight chain or branched alkyl having 1 to 7    carbon atoms or straight chain or branched alkyloxy having 1 to 7    carbon atoms, each of which optionally has 1 to 3 substituents    selected from the group consisting of a halogen atom, cyano,    hydroxy, halogenated alkyl and alkyloxy,-   provided that each of R² and R⁴ bonded to each other, R³ and R⁶    bonded to each other, R⁶ and R⁷ bonded to each other, and R⁷ and R⁸    bonded to each other may independently forms a ring optionally    having 1 to 3 substituents selected from the group consisting of a    halogen atom; cyano; hydroxy; amino; mono-substituted amino;    di-substituted amino; halogenated alkyl; alkylsulfanyl;    benzimidazolonyl; and straight chain or branched alkyl having 1 to 5    carbon atoms or straight chain or branched alkyloxy having 1 to 5    carbon atoms, each of which optionally has 1 to 3 substituents    selected from the group consisting of a halogen atom, cyano,    hydroxy, amino, mono-substituted amino, di-substituted amino,    alkyloxy and alkylsulfanyl.-   [3] A compound capable of binding to an NCS protein, which is    selected from the group consisting of the following formulas (1′) to    (11′), or a salt thereof; a compound represented by the formula    (1′):

wherein

-   Bridge¹′ is a bridge structure selected from the group consisting of    the following formulas (1a′) to (1j′):

-   R^(1a)′ is phenyl optionally having 1 to 3 substituents selected    from the group consisting of straight chain or branched alkyl having    1 to 9 carbon atoms, a halogen atom, cyano, hydroxy and amino; or    phenyl substituted by R^(4a)′,-   R^(2a)′ is straight chain or branched alkyl having 1 to 9 carbon    atoms; phenyl, cycloalkyl having 3 to 7 carbon atoms, phenylalkyl    having 7 to 11 carbon atoms, imidazolyl, biphenyl, thienyl,    benzothienyl or benzofuryl, each of which optionally has 1 to 3    substituents selected from the group consisting of straight chain or    branched alkyl having 1 to 9 carbon atoms, a halogen atom, cyano,    hydroxy, amino, mono-substituted amino, di-substituted amino,    alkylsulfanyl and halogenated methyl; or phenyl, imidazolyl,    biphenyl, thienyl, benzothienyl or benzofuryl, each of which is    substituted by R^(5a)′,-   R^(3a)′ is a hydrogen atom; straight chain or branched alkyl having    1 to 5 carbon atoms, cycloalkyl having 3 to 7 carbon atoms, phenyl,    biphenyl, piperidinyl, piperazinyl, imidazolyl, benzimidazolonyl or    morpholinyl, each of which optionally has 1 to 3 substituents    selected from the group consisting of a straight chain or branched    alkyl group having 1 to 9 carbon atoms, a halogen atom, cyano,    hydroxy, methoxy, amino, mono-substituted amino, di-substituted    amino, alkylsulfanyl and halogenated methyl; or a straight chain or    branched alkyl group having 1 to 5 carbon atoms, cycloalkyl having 3    to 7 carbon atoms, phenyl, biphenyl, piperidinyl, piperazinyl,    imidazolyl, benzimidazolonyl or morpholinyl, each of which is    substituted by R^(6a)′, and-   X′ is a hydrogen atom or straight chain or branched alkyl having 1    to 5 carbon atoms,-   provided that any one of R^(0a)′, R^(4a)′, R^(5a)′ and R^(6a)′ is    optionally a group selected from the group consisting of the    formulas (1B) to (1D):

wherein X^(0a)′ is straight chain or branched alkylene having 1 to 5carbon atoms;a compound represented by the formula (2′):

wherein

-   Bridge²′ is a bridge structure selected from the group consisting of    the following formulas (2a′) to (2r′):

wherein X¹′ is straight chain or branched alkylene having 1 to 5 carbonatoms, straight chain or branched alkenylene having 2 to 5 carbon atomsor straight chain or branched alkynylene having 2 to 5 carbon atoms, andX²′ and X³ are each independently straight chain or branched alkylenehaving 1 to 3 carbon atoms, straight chain or branched alkenylene having2 to 3 carbon atoms or alkynylene having 2 to 3 carbon atoms,

-   R^(1b)′ is phenyl optionally having 1 to 3 substituents selected    from the group consisting of straight chain or branched alkyl having    1 to 9 carbon atoms, a halogen atom, cyano, hydroxy and amino; or    phenyl substituted by R^(4b)′,-   R^(2b)′ is straight chain or branched alkyl having 1 to 9 carbon    atoms; phenyl, cycloalkyl having 3 to 7 carbon atoms, phenylalkyl    having 7 to 11 carbon atoms, imidazolyl, biphenyl, thienyl,    benzothienyl or benzofuryl, each of which optionally has 1 to 3    substituents selected from the group consisting of straight chain or    branched alkyl having 1 to 9 carbon atoms, a halogen atom, cyano,    hydroxy, amino, mono-substituted amino, di-substituted amino,    alkylsulfanyl and halogenated methyl; or phenyl, imidazolyl,    biphenyl, thienyl, benzothienyl or benzofuryl, each of which is    substituted by R^(5b)′, and-   R^(3b)′ is a hydrogen atom; straight chain or branched alkyl having    1 to 5 carbon atoms, cycloalkyl having 3 to 7 carbon atoms, phenyl,    biphenyl, piperidinyl, piperazinyl, imidazolyl, benzimidazolonyl or    morpholinyl, each of which optionally has 1 to 3 substituents    selected from the group consisting of a straight chain or branched    alkyl group having 1 to 9 carbon atoms, a halogen atom, cyano,    hydroxy, methoxy, amino, mono-substituted amino, di-substituted    amino, alkylsulfanyl and halogenated methyl; or a straight chain or    branched alkyl group having 1 to 5 carbon atoms, cycloalkyl having 3    to 7 carbon atoms, phenyl, biphenyl, piperidinyl, piperazinyl,    imidazolyl, benzimidazolonyl or morpholinyl, each of which is    substituted by R^(6b)′,-   provided that any one of R^(4b)′, R^(5b)′ and R^(6b)′ is optionally    a group selected from the group consisting of the formulas (2B) to    (2D):

wherein X^(0b)′ is straight chain or branched alkylene having 1 to 5carbon atoms;a compound represented by the formula (3′):

wherein

-   Bridge³′ is a bridge structure selected from the group consisting of    the following formulas (3a′) to (3f′):

wherein X⁴ is straight chain or branched alkylene having 1 to 5 carbonatoms, and R^(7c)′ is straight chain or branched alkyl having 1 to 5carbon atoms,

-   R^(1c)′ is phenyl optionally having 1 to 3 substituents selected    from the group consisting of straight chain or branched alkyl having    1 to 9 carbon atoms, a halogen atom, cyano, hydroxy and amino; or    phenyl substituted by R^(4c)′,-   R²c′ is straight chain or branched alkyl having 1 to 9 carbon atoms;    phenyl, cycloalkyl having 3 to 7 carbon atoms, phenylalkyl having 7    to 11 carbon atoms, imidazolyl, biphenyl, thienyl, benzothienyl or    benzofuryl, each of which optionally has 1 to 3 substituents    selected from the group consisting of straight chain or branched    alkyl having 1 to 9 carbon atoms, a halogen atom, cyano, hydroxy,    amino, mono-substituted amino, di-substituted amino, alkylsulfanyl    and halogenated methyl; or phenyl, imidazolyl, biphenyl, thienyl,    benzothienyl or benzofuryl, each of which is substituted by R^(5c)′,    and-   R^(3c)′ is a hydrogen atom; straight chain or branched alkyl having    1 to 5 carbon atoms, cycloalkyl having 3 to 7 carbon atoms, phenyl,    biphenyl, pyrrolidinyl, piperidinyl, piperazinyl, imidazolyl,    benzimidazolonyl or morpholinyl, each of which optionally has 1 to 3    substituents selected from the group consisting of a straight chain    or branched alkyl group having 1 to 9 carbon atoms, a halogen atom,    cyano, hydroxy, methoxy, amino, mono-substituted amino,    di-substituted amino, alkylsulfanyl and halogenated methyl; or a    straight chain or branched alkyl group having 1 to 5 carbon atoms,    cycloalkyl having 3 to 7 carbon atoms, phenyl, biphenyl,    pyrrolidinyl, piperidinyl, piperazinyl, imidazolyl, benzimidazolonyl    or morpholinyl, each of which is substituted by R^(6c)′,-   provided that any one of R^(0c)′, R^(4c)′, R^(5c)′, R^(6c)′ and    R^(7c)′ is optionally a group selected from the group consisting of    the formulas (3B) to (3D):

wherein X^(0c)′ is straight chain or branched alkylene having 1 to 5carbon atoms;a compound represented by the formula (4′):

wherein

-   R^(1d)′ is phenyl optionally having 1 to 3 substituents selected    from the group consisting of straight chain or branched alkyl having    1 to 9 carbon atoms, a halogen atom, cyano, hydroxy and amino; or    phenyl substituted by R^(4d)′,-   R^(2d)′ is straight chain or branched alkyl having 1 to 9 carbon    atoms; or phenyl, cycloalkyl having 3 to 7 carbon atoms, phenylalkyl    having 7 to 11 carbon atoms, imidazolyl, biphenyl, thienyl,    benzothienyl or benzofuryl, each of which optionally has 1 to 3    substituents selected from the group consisting of straight chain or    branched alkyl having 1 to 9 carbon atoms, a halogen atom, cyano,    hydroxy, amino, mono-substituted amino, di-substituted amino,    alkylsulfanyl, halogenated methyl and R^(5d)′,-   R^(4d)′ is optionally a group represented by the formula (d1′):

wherein X⁵′ is straight chain or branched alkylene having 1 to 5 carbonatoms, and

-   R^(5d)′ is 4-hydroxyphenyl,-   provided that any one of R^(4d)′ and R^(5d)′ is optionally a group    selected from the group consisting of the formulas (4B) to (4D):

wherein X^(0d)′ is straight chain or branched alkylene having 1 to 5carbon atoms;a compound represented by the formula (5′):

wherein

-   Bridge⁵′ is a bridge structure represented by the following formula    (5a′):

wherein R^(2e)′ is benzene, cycloalkane having 3 to 7 carbon atoms,imidazole, biphenyl, thiophene, benzothiophene or benzofuran, each ofwhich optionally has 1 to 3 substituents selected from the groupconsisting of straight chain or branched alkyl having 1 to 9 carbonatoms, a halogen atom, cyano, hydroxy, amino, mono-substituted amino,di-substituted amino, alkylsulfanyl and halogenated methyl; or benzene,imidazole, biphenyl, thiophene, benzothiophene or benzofuran, each ofwhich is substituted by R^(5e)′,

-   R^(1e)′ is phenyl optionally having 1 to 3 substituents selected    from the group consisting of straight chain or branched alkyl having    1 to 9 carbon atoms, a halogen atom, cyano, hydroxy and amino; or    phenyl substituted by R^(4e)′, and-   R^(3e)′ is a hydrogen atom; straight chain or branched alkyl having    1 to 5 carbon atoms, cycloalkyl having 3 to 7 carbon atoms, phenyl,    biphenyl, piperidinyl, piperazinyl, imidazolyl, benzimidazolonyl or    morpholinyl, each of which optionally has 1 to 3 substituents    selected from the group consisting of a straight chain or branched    alkyl group having 1 to 9 carbon atoms, a halogen atom, cyano,    hydroxy, methoxy, amino, mono-substituted amino, di-substituted    amino, alkylsulfanyl and halogenated methyl; or a straight chain or    branched alkyl group having 1 to 5 carbon atoms, cycloalkyl having 3    to 7 carbon atoms, phenyl, biphenyl, piperidinyl, piperazinyl,    imidazolyl, benzimidazolonyl or morpholinyl, each of which is    substituted by R^(6e)′,-   provided that any one of R^(3e)′, R^(4e)′, R^(5e)′ and R^(6e)′ is    optionally a group selected from the group consisting of the    formulas (5B) to (5D):

wherein X^(0e)′ is straight chain or branched alkylene having 1 to 5carbon atoms;a compound represented by the formula (6′):

wherein

-   Bridge⁶′ is a bridge structure selected from the group consisting of    the following formulas (6a′) to (6g′):

wherein X⁶′ and X⁹′ are each independently straight chain or branchedalkylene having 1 to 5 carbon atoms, and X⁷′, X⁸′, X¹⁰′ and X¹¹′ areeach independently straight chain or branched alkylene having 1 to 3carbon atoms,

-   R^(1f)′ is phenyl optionally having 1 to 3 substituents selected    from the group consisting of straight chain or branched alkyl having    1 to 9 carbon atoms, a halogen atom, cyano, hydroxy and amino; or    phenyl substituted by R^(4f)′, and-   R^(2f)′ is straight chain or branched alkyl having 1 to 9 carbon    atoms; phenyl, cycloalkyl having 3 to 7 carbon atoms, phenylalkyl    having 7 to 11 carbon atoms, imidazolyl, biphenyl, thienyl,    benzothienyl or benzofuryl, each of which optionally has 1 to 3    substituents selected from the group consisting of straight chain or    branched alkyl having 1 to 9 carbon atoms, a halogen atom, cyano,    hydroxy, amino, mono-substituted amino, di-substituted amino,    alkylsulfanyl and halogenated methyl; a group represented by the    formula (f1′):

-   or phenyl, imidazolyl, biphenyl, thienyl, benzothienyl or    benzofuryl, each of which substituted by R^(5f)′,-   provided that any one of R^(4f)′ and R^(5f)′ is optionally a group    selected from the group consisting of the formulas (6B) to (6D):

wherein X^(0f)′ is straight chain or branched alkylene having 1 to 5carbon atoms;a compound represented by the formula (7′):

wherein

-   Bridge⁷′ is a bridge structure represented by the following formula    (7a′):

wherein X¹²′ is straight chain or branched alkylene having 1 to 5 carbonatoms,

-   R^(1g)′ is phenyl optionally having 1 to 3 substituents selected    from the group consisting of straight chain or branched alkyl having    1 to 9 carbon atoms, a halogen atom, cyano, hydroxy and amino; or    phenyl substituted by R^(4g)′,-   R^(2g)′ is divalent pyridazinyl optionally having 1 to 3    substituents selected from the group consisting of straight chain or    branched alkyl having 1 to 9 carbon atoms, a halogen atom, cyano,    hydroxy, amino, mono-substituted amino, di-substituted amino,    alkylsulfanyl and halogenated methyl; or divalent pyridazinyl    substituted by R^(5g)′, and-   R^(3g)′ is a hydrogen atom; straight chain or branched alkyl having    1 to 5 carbon atoms, cycloalkyl having 3 to 7 carbon atoms, phenyl,    biphenyl, piperidinyl, piperazinyl, imidazolyl, benzimidazolonyl or    morpholinyl, each of which optionally has 1 to 3 substituents    selected from the group consisting of a straight chain or branched    alkyl group having 1 to 9 carbon atoms, a halogen atom, cyano,    hydroxy, methoxy, amino, mono-substituted amino, di-substituted    amino, alkylsulfanyl and halogenated methyl, or a straight chain or    branched alkyl group having 1 to 5 carbon atoms, cycloalkyl having 3    to 7 carbon atoms, phenyl, biphenyl, piperidinyl, piperazinyl,    imidazolyl, benzimidazolonyl or morpholinyl, each of which is    substituted by R^(6g)′,-   provided that any one of R^(3g)′, R^(4g)′, R^(5g)′ and R^(6g)′ is    optionally a group selected from the group consisting of the    formulas (7B) to (7D):

wherein X^(0g)′ is straight chain or branched alkylene having 1 to 5carbon atoms;a compound selected from the group consisting of the following formulas(8a′) to (8j′):

wherein

-   X¹³′ is straight chain or branched alkylene having 1 to 5 carbon    atoms,-   R^(3h)′ is a hydrogen atom; straight chain or branched alkyl having    1 to 5 carbon atoms, cycloalkyl having 3 to 7 carbon atoms, phenyl,    biphenyl, piperidinyl, piperazinyl, imidazolyl, benzimidazolonyl or    morpholinyl, each of which optionally has 1 to 3 substituents    selected from the group consisting of a straight chain or branched    alkyl group having 1 to 9 carbon atoms and optionally having    hydroxy, a halogen atom, cyano, hydroxy, methoxy, amino,    mono-substituted amino, di-substituted amino, alkylsulfanyl and    halogenated methyl; or a straight chain or branched alkyl group    having 1 to 5 carbon atoms, cycloalkyl having 3 to 7 carbon atoms,    phenyl, biphenyl, piperidinyl, piperazinyl, imidazolyl,    benzimidazolonyl or morpholinyl, each of which is substituted by    R^(6h)′, and-   R^(7h)′ is a hydrogen atom; phenothiazinyl, phenazinyl,    dihydrophenazinyl, thioxanthenyl, dibenzoxazepinyl, phenoxazinyl,    acrydinyl, xanthenyl, thianthrenyl or phenoxathiinyl, each of which    optionally has 1 to 3 substituents selected from the group    consisting of a straight chain or branched alkyl group having 1 to 9    carbon atoms, a halogen atom, cyano, hydroxy, amino,    mono-substituted amino, di-substituted amino, substituted imino,    alkylsulfanyl and halogenated methyl; or a straight chain or    branched alkyl group having 1 to 5 carbon atoms, phenothiazinyl    having 3 to 7 carbon atoms, phenazinyl, dihydrophenazinyl,    thioxanthenyl, dibenzoxazepinyl, phenoxazinyl, acrydinyl, xanthenyl,    thianthrenyl or phenoxathiinyl, each of which is substituted by    R^(5h)′,-   provided that any one of R^(3h)′, R^(5h)′ and R^(6h)′ is optionally    a group selected from the group consisting of the formulas (8B) to    (8D):

wherein X^(0h)′ is straight chain or branched alkylene having 1 to 5carbon atoms;a compound represented by the formula (9′):

wherein

-   Bridge⁹′ is a bridge structure selected from the group consisting of    the formulas (9a′) and (9b′):

-   R^(8i)′ is a group represented by the formula (i1′):

-   R^(9i)′ is a hydrogen atom; straight chain or branched alkyl having    1 to 5 carbon atoms, cycloalkyl having 3 to 7 carbon atoms, phenyl,    biphenyl, piperidinyl, piperazinyl, imidazolyl, benzimidazolonyl or    morpholinyl, each of which optionally has 1 to 3 substituents    selected from the group consisting of a straight chain or branched    alkyl group having 1 to 9 carbon atoms, a halogen atom, cyano,    hydroxy, methoxy, alkoxycarbonyloxy, amino, mono-substituted amino,    di-substituted amino, alkylsulfanyl and halogenated methyl; or a    straight chain or branched alkyl group having 1 to 5 carbon atoms,    cycloalkyl having 3 to 7 carbon atoms, phenyl, biphenyl,    piperidinyl, piperazinyl, imidazolyl, benzimidazolonyl or    morpholinyl, each of which is substituted by R^(6i)′,-   provided that any one of R^(6i)′ and R^(9i)′ is optionally a group    selected from the group consisting of the formulas (9B) to (9D):

wherein X^(0i)′ is straight chain or branched alkylene having 1 to 5carbon atoms;a compound represented by the formula (10′):

wherein

-   Bridge¹⁰′ is a bridge structure selected from the group consisting    of the formulas (10a′) and (10b′):

-   R^(10j)′ is a group represented by the formula (j1′):

-   R^(11j)′ is a group represented by the formula (j2′)

wherein X¹⁴′ is isopropyl, isobutyl, sec-butyl or benzyl; straight chainor branched alkyl having 1 to 5 carbon atoms, cycloalkyl having 3 to 7carbon atoms, phenyl, biphenyl, piperidinyl, piperazinyl, imidazolyl,benzimidazolonyl or morpholinyl, each of which optionally has 1 to 3substituents selected from the group consisting of a straight chain orbranched alkyl group having 1 to 9 carbon atoms, a halogen atom, cyano,hydroxy, methoxy, amino, mono-substituted amino, di-substituted amino,alkylsulfanyl and halogenated methyl; or a straight chain or branchedalkyl group having 1 to 5 carbon atoms, cycloalkyl having 3 to 7 carbonatoms, phenyl, biphenyl, piperidinyl, piperazinyl, imidazolyl,benzimidazolonyl or morpholinyl, each of which is substituted byR^(6j)′,

-   provided that any one of R^(6j)′ and R^(11j)′ is optionally a group    selected from the group consisting of the formulas (10B) to (10D):

wherein X^(0j)′ is straight chain or branched alkylene having 1 to 5carbon atoms; anda compound represented by the formula (11′):

wherein

-   R^(12k)′ is a group selected from the group consisting of the    formulas (11a′) and (11b′):

-   R^(13k)′ is a hydrogen atom; straight chain or branched alkyl having    1 to 5 carbon atoms, cycloalkyl having 3 to 7 carbon atoms, phenyl,    biphenyl, piperidinyl, piperazinyl, imidazolyl, benzimidazolonyl or    morpholinyl, each of which optionally has 1 to 3 substituents    selected from the group consisting of a straight chain or branched    alkyl group having 1 to 9 carbon atoms, a halogen atom, cyano,    hydroxy, methoxy, amino, mono-substituted amino, di-substituted    amino, alkylsulfanyl and halogenated methyl; or a straight chain or    branched alkyl group having 1 to 5 carbon atoms, cycloalkyl having 3    to 7 carbon atoms, phenyl, biphenyl, piperidinyl, piperazinyl,    imidazolyl, benzimidazolonyl or morpholinyl, each of which is    substituted by R^(6k)′,-   provided that any one of R^(6k)′ and R^(13k)′ is optionally a group    selected from the group consisting of the formulas (11B) to (11D):

wherein X^(0k)′ is straight chain or branched alkylene having 1 to 5carbon atoms.

-   [4] A drug for the treatment or prophylaxis of dementia, which    comprises a compound selected from the group consisting of the    following formulas (1) to (11), or a pharmaceutically acceptable    salt thereof, as an active ingredient;    a compound represented by the formula (1):

wherein

-   Bridge¹ is a bridge structure selected from the group consisting of    the following formulas (1a) to (1j):

-   R^(1a) is phenyl optionally having 1 to 3 substituents selected from    the group consisting of straight chain or branched alkyl having 1 to    9 carbon atoms, a halogen atom, cyano, hydroxy and amino,-   R^(2a) is straight chain or branched alkyl having 1 to 9 carbon    atoms; or phenyl, cycloalkyl having 3 to 7 carbon atoms, phenylalkyl    having 7 to 11 carbon atoms, imidazolyl, biphenyl, thienyl,    benzothienyl or benzofuryl, each of which optionally has 1 to 3    substituents selected from the group consisting of straight chain or    branched alkyl having 1 to 9 carbon atoms, a halogen atom, cyano,    hydroxy, amino, mono-substituted amino, di-substituted amino,    alkylsulfanyl and halogenated methyl,-   R^(3a) is a hydrogen atom; or straight chain or branched alkyl    having 1 to 5 carbon atoms, cycloalkyl having 3 to 7 carbon atoms,    phenyl, biphenyl, piperidinyl, piperazinyl, imidazolyl,    benzimidazolonyl or morpholinyl, each of which optionally has 1 to 3    substituents selected from the group consisting of a straight chain    or branched alkyl group having 1 to 9 carbon atoms, a halogen atom,    cyano, hydroxy, methoxy, amino, mono-substituted amino,    di-substituted amino, alkylsulfanyl and halogenated methyl, and-   X is a hydrogen atom or straight chain or branched alkyl having 1 to    5 carbon atoms;    a compound represented by the formula (2):

wherein

-   Bridge² is a bridge structure selected from the group consisting of    the following formulas (2a) to (2r):

wherein X¹ is straight chain or branched alkylene having 1 to 5 carbonatoms, straight chain or branched alkenylene having 2 to 5 carbon atomsor straight chain or branched alkynylene having 2 to 5 carbon atoms, andX² and X³ are each independently straight chain or branched alkylenehaving 1 to 3 carbon atoms, straight chain or branched alkenylene having2 to 3 carbon atoms or alkynylene having 2 to 3 carbon atoms,

-   R^(1b) is phenyl optionally having 1 to 3 substituents selected from    the group consisting of straight chain or branched alkyl having 1 to    9 carbon atoms, a halogen atom, cyano, hydroxy and amino,-   R^(2b) is straight chain or branched alkyl having 1 to 9 carbon    atoms; or phenyl, cycloalkyl having 3 to 7 carbon atoms, phenylalkyl    having 7 to 11 carbon atoms, imidazolyl, biphenyl, thienyl,    benzothienyl or benzofuryl, each of which optionally has 1 to 3    substituents selected from the group consisting of straight chain or    branched alkyl having 1 to 9 carbon atoms, a halogen atom, cyano,    hydroxy, amino, mono-substituted amino, di-substituted amino,    alkylsulfanyl and halogenated methyl, and-   R^(3b) is a hydrogen atom; or straight chain or branched alkyl    having 1 to 5 carbon atoms, cycloalkyl having 3 to 7 carbon atoms,    phenyl, biphenyl, piperidinyl, piperazinyl, imidazolyl,    benzimidazolonyl or morpholinyl, each of which optionally has 1 to 3    substituents selected from the group consisting of a straight chain    or branched alkyl group having 1 to 9 carbon atoms, a halogen atom,    cyano, hydroxy, methoxy, amino, mono-substituted amino,    di-substituted amino, alkylsulfanyl and halogenated methyl;    a compound represented by the formula (3):

wherein

-   Bridge³ is a bridge structure selected from the group consisting of    the following formulas (3a) to (3f):

wherein X⁴ is straight chain or branched alkylene having 1 to 5 carbonatoms, and R^(7c) is a straight chain or branched alkyl group having 1to 5 carbon atoms,

-   R^(1c) is phenyl optionally having 1 to 3 substituents selected from    the group consisting of straight chain or branched alkyl having 1 to    9 carbon atoms, a halogen atom, cyano, hydroxy and amino,-   R^(2c) is straight chain or branched alkyl having 1 to 9 carbon    atoms; or phenyl, cycloalkyl having 3 to 7 carbon atoms, phenylalkyl    having 7 to 11 carbon atoms, imidazolyl, biphenyl, thienyl,    benzothienyl or benzofuryl, each of which optionally has 1 to 3    substituents selected from the group consisting of straight chain or    branched alkyl having 1 to 9 carbon atoms, a halogen atom, cyano,    hydroxy, amino, mono-substituted amino, di-substituted amino,    alkylsulfanyl and halogenated methyl, and-   R^(3c) is a hydrogen atom; or straight chain or branched alkyl    having 1 to 5 carbon atoms, cycloalkyl having 3 to 7 carbon atoms,    phenyl, biphenyl, pyrrolidinyl, piperidinyl, piperazinyl,    imidazolyl, benzimidazolonyl or morpholinyl, each of which    optionally has 1 to 3 substituents selected from the group    consisting of a straight chain or branched alkyl group having 1 to 9    carbon atoms, a halogen atom, cyano, hydroxy, methoxy, amino,    mono-substituted amino, di-substituted amino, alkylsulfanyl and    halogenated methyl;    a compound represented by the formula (4):

wherein R^(1d) is phenyl optionally having 1 to 3 substituents selectedfrom the group consisting of straight chain or branched alkyl having 1to 9 carbon atoms, a halogen atom, cyano, hydroxy and amino,

-   R^(2d) is straight chain or branched alkyl having 1 to 9 carbon    atoms; or phenyl, cycloalkyl having 3 to 7 carbon atoms, phenylalkyl    having 7 to 11 carbon atoms, imidazolyl, biphenyl, thienyl,    benzothienyl or benzofuryl, each of which optionally has 1 to 3    substituents selected from the group consisting of straight chain or    branched alkyl having 1 to 9 carbon atoms, a halogen atom, cyano,    hydroxy, amino, mono-substituted amino, di-substituted amino,    alkylsulfanyl, halogenated methyl and R^(5d), R^(4d) is a group    represented by the formula (d1):

wherein X⁵ is straight chain or branched alkylene having 1 to 5 carbonatoms, and

-   R^(5d) is 4-hydroxyphenyl;    a compound represented by the formula (5):

wherein

-   Bridge⁵ is a bridge structure represented by the following formula    (5a):

wherein R^(2e) is benzene, cycloalkane having 3 to 7 carbon atoms,imidazole, biphenyl, thiophene, benzothiophene or benzofuran, each ofwhich optionally has 1 to 3 substituents selected from the groupconsisting of straight chain or branched alkyl having 1 to 9 carbonatoms, a halogen atom, cyano, hydroxy, amino, mono-substituted amino,di-substituted amino, alkylsulfanyl and halogenated methyl,

-   R^(1e) is phenyl optionally having 1 to 3 substituents selected from    the group consisting of straight chain or branched alkyl having 1 to    9 carbon atoms, a halogen atom, cyano, hydroxy and amino, and-   R^(3e) is a hydrogen atom; or straight chain or branched alkyl    having 1 to 5 carbon atoms, cycloalkyl having 3 to 7 carbon atoms,    phenyl, biphenyl, piperidinyl, piperazinyl, imidazolyl,    benzimidazolonyl or morpholinyl, each of which optionally has 1 to 3    substituents selected from the group consisting of a straight chain    or branched alkyl group having 1 to 9 carbon atoms, a halogen atom,    cyano, hydroxy, methoxy, amino, mono-substituted amino,    di-substituted amino, alkylsulfanyl and halogenated methyl;    a compound represented by the formula (6):

wherein

-   Bridge⁶ is a bridge structure selected from the group consisting of    the following formulas (6a) to (6g):

wherein X⁶ and X⁹ are each independently straight chain or branchedalkylene having 1 to 5 carbon atoms, and X⁷, X⁸, X¹⁰ and X¹¹ are eachindependently straight chain or branched alkylene having 1 to 3 carbonatoms,

-   R^(1f) is phenyl optionally having 1 to 3 substituents selected from    the group consisting of straight chain or branched alkyl having 1 to    9 carbon atoms, a halogen atom, cyano, hydroxy and amino, and-   R^(2f) is straight chain or branched alkyl having 1 to 9 carbon    atoms; phenyl, cycloalkyl having 3 to 7 carbon atoms, phenylalkyl    having 7 to 11 carbon atoms, imidazolyl, biphenyl, thienyl,    benzothienyl or benzofuryl, each of which optionally has 1 to 3    substituents selected from the group consisting of straight chain or    branched alkyl having 1 to 9 carbon atoms, a halogen atom, cyano,    hydroxy, amino, mono-substituted amino, di-substituted amino,    alkylsulfanyl and halogenated methyl; or a group represented by the    formula (f1):

a compound represented by the formula (7):

wherein

-   Bridge⁷ is a bridge structure represented by the following formula    (7a):

wherein X¹² is straight chain or branched alkylene having 1 to 5 carbonatoms,

-   R^(1g) is phenyl optionally having 1 to 3 substituents selected from    the group consisting of straight chain or branched alkyl having 1 to    9 carbon atoms, a halogen atom, cyano, hydroxy and amino,-   R^(2g) is divalent pyridazinyl optionally having 1 to 3 substituents    selected from the group consisting of straight chain or branched    alkyl having 1 to 9 carbon atoms, a halogen atom, cyano, hydroxy,    amino, mono-substituted amino, di-substituted amino, alkylsulfanyl    and halogenated methyl, and-   R^(3g) is a hydrogen atom; or straight chain or branched alkyl    having 1 to 5 carbon atoms, cycloalkyl having 3 to 7 carbon atoms,    phenyl, biphenyl, piperidinyl, piperazinyl, imidazolyl,    benzimidazolonyl or morpholinyl, each of which optionally has 1 to 3    substituents selected from the group consisting of a straight chain    or branched alkyl group having 1 to 9 carbon atoms, a halogen atom,    cyano, hydroxy, methoxy, amino, mono-substituted amino,    di-substituted amino, alkylsulfanyl and halogenated methyl;    a compound selected from the group consisting of represented by the    following formulas (8a) to (8j):

wherein

-   X¹³ is straight chain or branched alkylene having 1 to 5 carbon    atoms,-   R^(3h) is a hydrogen atom; or straight chain or branched alkyl    having 1 to 5 carbon atoms, cycloalkyl having 3 to 7 carbon atoms,    phenyl, biphenyl, piperidinyl, piperazinyl, imidazolyl,    benzimidazolonyl or morpholinyl, each of which optionally has 1 to 3    substituents selected from the group consisting of a straight chain    or branched alkyl group having 1 to 9 carbon atoms and optionally    having hydroxy, a halogen atom, cyano, hydroxy, methoxy, amino,    mono-substituted amino, di-substituted amino, alkylsulfanyl and    halogenated methyl, and-   R^(7h) is a hydrogen atom; or phenothiazinyl, phenazinyl,    dihydrophenazinyl, thioxanthenyl, dibenzoxazepinyl, phenoxazinyl,    acrydinyl, xanthenyl, thianthrenyl or phenoxathiinyl, each of which    optionally has 1 to 3 substituents selected from the group    consisting of a straight chain or branched alkyl group having 1 to 9    carbon atoms, a halogen atom, cyano, hydroxy, amino,    mono-substituted amino, di-substituted amino, substituted imino,    alkylsulfanyl and halogenated methyl;    a compound represented by the formula (9):

wherein

-   Bridge⁹ is a bridge structure selected from the group consisting of    the formulas (9a) to (9b):

-   R^(8i) is a group represented by the formula (i1):

-   R^(9i) is a hydrogen atom; or straight chain or branched alkyl    having 1 to 5 carbon atoms, cycloalkyl having 3 to 7 carbon atoms,    phenyl, biphenyl, piperidinyl, piperazinyl, imidazolyl,    benzimidazolonyl or morpholinyl, each of which optionally has 1 to 3    substituents selected from the group consisting of a straight chain    or branched alkyl group having 1 to 9 carbon atoms, a halogen atom,    cyano, hydroxy, methoxy, alkoxycarbonyloxy, amino, mono-substituted    amino, di-substituted amino, alkylsulfanyl and halogenated methyl;    a compound represented by the formula (10):

wherein

-   Bridge¹⁰ is a bridge structure selected from the group consisting of    the formulas (10a) and (10b):

-   R^(10j) is a group represented by the formula (j1):

-   R^(11j) is a group represented by the formula (j2)

wherein X¹⁴ is isopropyl, isobutyl, sec-butyl or benzyl; or straightchain or branched alkyl having 1 to 5 carbon atoms, cycloalkyl having 3to 7 carbon atoms, phenyl, biphenyl, piperidinyl, piperazinyl,imidazolyl, benzimidazolonyl or morpholinyl, each of which optionallyhas 1 to 3 substituents selected from the group consisting of a straightchain or branched alkyl group having 1 to 9 carbon atoms, a halogenatom, cyano, hydroxy, methoxy, amino, mono-substituted amino,di-substituted amino, alkylsulfanyl and halogenated methyl; anda compound represented by the formula (11):

wherein

-   R^(12k) is a group selected from the group consisting of the    formulas (11a) and (11b):

-   R^(13k) is a hydrogen atom; or straight chain or branched alkyl    having 1 to 5 carbon atoms, cycloalkyl having 3 to 7 carbon atoms,    phenyl, biphenyl, piperidinyl, piperazinyl, imidazolyl,    benzimidazolonyl or morpholinyl, each of which optionally has 1 to 3    substituents selected from the group consisting of a straight chain    or branched alkyl group having 1 to 9 carbon atoms, a halogen atom,    cyano, hydroxy, methoxy, amino, mono-substituted amino,    di-substituted amino, alkylsulfanyl and halogenated methyl.-   [5] A method for screening a drug, which comprises evaluating    whether or not a test substance is capable of regulating the    expression or function of an NCS protein gene.-   [6] The method according to the aforementioned [5], wherein the drug    is an agent of regulating central nervous action, dementia action or    Alzheimer's disease action.-   [7] The method according to the aforementioned [5], wherein the drug    is a substance capable of regulating an action associated with an    NCS protein-targeting drug.-   [8] The method according to the aforementioned [5], wherein the NCS    protein gene is a neurocalcin gene.-   [9] The method according to the aforementioned [5], wherein the NCS    protein gene is a neurocalcin 5 gene.-   [10] The method according to the aforementioned [5], which comprises    the following steps (a) to (c):-   (a) a step for bringing the test substance into contact with the NCS    protein or mutant protein thereof;-   (b) a step for measuring the functional level of the protein or    mutant protein thereof in the presence of the test substance, and    comparing said functional level with the functional level of the    protein or mutant protein thereof in the absence of the test    substance;-   (c) a step for selecting a test substance that alters the functional    level of the protein or mutant protein thereof on the basis of the    result of the comparison in (b) above.-   [11] The method according to the aforementioned [5], which comprises    the following steps (a) to (c):-   (a) a step for bringing the test substance into contact with cells    enabling a measurement of the expression of the NCS protein or a    gene encoding the protein;-   (b) a step for measuring the expression level of the NCS protein or    the gene in the cells in contact with the test substance, and    comparing said expression level with the expression level of the    protein or the gene in control cells not in contact with the test    substance;-   (c) a step for selecting a test substance that regulates the    expression level of the protein or the gene on the basis of the    result of the comparison in step (b) above.-   [12] The method according to the aforementioned [5], which comprises    the following steps (a) to (c):-   (a) a step for bringing the test substance into contact with an NCS    protein or a mutant protein thereof;-   (b) a step for measuring the ability of the test substance to bind    to said protein;-   (c) a step for selecting a test substance that has the binding    ability to said protein, on the basis of the result of step (b)    above.-   [13] The method according to the aforementioned [5], which comprises    the following steps (a) to (c):-   (a) a step for bringing the test substance and an NCS    protein-binding substance into contact with an NCS protein or a    mutant protein thereof;-   (b) a step for measuring the binding amount of the NCS    protein-binding substance to said protein in the presence of the    test substance, and comparing said amount with the binding amount of    the NCS protein-binding substance to said protein in the absence of    the test substance;-   (c) a step for selecting a test substance that alters the binding    amount of the NCS protein-binding substance to said protein on the    basis of the result of the comparison in step (b) above.-   [14] The method according to the aforementioned [13], wherein the    NCS protein-binding substance is atorvastatin, pimozide, bifonazole,    flunarizine, fendiline, chloperastine, bepridil, raloxifene    hydrochloride, benzbromarone, prazepam, clotiazepam, suloctidil,    benzethonium, bicaltamide, benzthiazide, minaprine, trifluoperazine,    chlorprothixene, pimethixene, flupentixol, clofazimine, loxapine,    rescinnamine, syrosingopine, dihydroergocornine mesylate,    dihydro-α-ergocryptine mesylate, dihydro-β-ergocryptine mesylate,    dihydroergocristine mesylate or stanozolol, or a derivative thereof.-   [15] A method for screening a substance capable of regulating a    function associated with an NCS protein gene, which comprises    determining whether or not a test substance is capable of regulating    the ability of an NCS protein-targeting drug to bind to NCS or a    mutant protein thereof.-   [16] The method according to the aforementioned [15], wherein the    NCS protein-targeting drug is atorvastatin, pimozide, bifonazole,    flunarizine, fendiline, chloperastine, bepridil, raloxifene    hydrochloride, benzbromarone, prazepam, clotiazepam, suloctidil,    benzethonium, bicaltamide, benzthiazide, minaprine, trifluoperazine,    chlorprothixene, pimethixene, flupentixol, clofazimine, loxapine,    rescinnamine, syrosingopine, dihydroergocornine mesylate,    dihydro-α-ergocryptine mesylate, dihydro-β-ergocryptine mesylate,    dihydroergocristine mesylate or stanozolol, or a derivative thereof    having the ability to bind to NCS.-   [17] The method according to the aforementioned [15], which    comprises the following steps (a) to (c):-   (a) a step for bringing the test substance and the NCS    protein-targeting drug into contact with the NCS or the mutant    protein thereof;-   (b) a step for measuring the binding amount of the NCS    protein-targeting drug to said protein in the presence of the test    substance, and comparing said binding amount with the binding amount    of the NCS target drug to said protein in the absence of the test    substance;-   (c) a step for selecting a test substance that alters the binding    amount of the NCS protein-targeting drug to said protein on the    basis of the result of the comparison in step (b) above.-   [18] A substance obtained by the method according to any of the    aforementioned [5]-[17].-   [19] An agent of regulating a pharmacologic action, comprising a    substance obtained by the method according to any of the    aforementioned [5]-[17].-   [20] An agent of regulating a pharmacologic action, which comprises    a substance that regulates the expression or function of an NCS    protein gene.-   [21] The agent according to the aforementioned [20], wherein the    pharmacologic action is an anti-central nervous disease action, an    anti-dementia action or an anti-Alzheimer's disease action.-   [22] The agent according to the aforementioned [20], which is an    agent of regulating an action associated with an NCS    protein-targeting drug.-   [23] The agent according to the aforementioned [20], wherein the NCS    protein gene is a neurocalcin gene.-   [24] The agent according to the aforementioned [20], wherein the NCS    protein gene is a neurocalcin 5 gene.-   [25] The agent according to the aforementioned [20], wherein the    substance that regulates the expression or function of the NCS    protein gene is a substance that suppresses the expression or    function of the NCS protein gene, which is either (i) or (ii):-   (i) a nucleic acid selected from a group consisting of NCS antisense    nucleic acid, NCS ribozyme, NCS decoy nucleic acid, NCS siRNA,    nucleic acid which encodes an NCS antibody, and nucleic acid which    encodes an NCS dominant negative mutant protein, or an expression    vector that comprises said nucleic acid; or-   (ii) a protein selected from a group consisting of NCS antibody, and    NCS dominant negative mutant protein.-   [26] An agent of regulating a pharmacologic action, comprising an    NCS protein, or an expression vector comprising a nucleic acid which    encodes the NCS protein.-   [27] An agent of regulating a function associated with an NCS    protein gene, comprising an NCS protein-targeting drug.-   [28] The agent according to the aforementioned [27], wherein the NCS    protein-targeting drug is atorvastatin, pimozide, bifonazole,    flunarizine, fendiline, chloperastine, bepridil, raloxifene    hydrochloride, benzbromarone, prazepam, clotiazepam, suloctidil,    benzethonium, bicaltamide, benzthiazide, minaprine, trifluoperazine,    chlorprothixene, pimethixene, flupentixol, clofazimine, loxapine,    rescinnamine, syrosingopine, dihydroergocornine mesylate,    dihydro-α-ergocryptine mesylate, dihydro-β-ergocryptine mesylate,    dihydroergocristine mesylate or stanozolol, or a derivative thereof    having the binding ability to NCS.-   [29] A method of producing a derivative of a drug, which comprises    derivatizing the drug so as to be able to regulate the function of    an NCS protein gene.-   [30] The method according to the aforementioned [29], wherein the    drug is a statin which has an anti-central nervous disease action,    an anti-dementia action or an anti-Alzheimer's disease action.-   [31] The method according to the aforementioned [29], wherein the    drug is atorvastatin, pimozide, bifonazole, flunarizine, fendiline,    chloperastine, bepridil, raloxifene hydrochloride, benzbromarone,    prazepam, clotiazepam, suloctidil, benzethonium, bicaltamide,    benzthiazide, minaprine, trifluoperazine, chlorprothixene,    pimethixene, flupentixol, clofazimine, loxapine, rescinnamine,    syrosingopine, dihydroergocornine mesylate, dihydro-α-ergocryptine    mesylate, dihydro-β-ergocryptine mesylate, dihydroergocristine    mesylate or stanozolol.-   [32] The method according to the aforementioned [29], wherein the    NCS protein gene is a neurocalcin gene.-   [33] The method according to the aforementioned [29], wherein the    NCS protein gene is a neurocalcin δ gene.-   [34] A method of producing a derivative of a substance capable of    regulating a function associated with an NCS protein gene, which    comprises derivatizing a drug so as to be able to regulate the    ability to bind to NCS or a mutant protein thereof.-   [35] The method according to the aforementioned [34], wherein the    drug is atorvastatin, pimozide, bifonazole, flunarizine, fendiline,    chloperastine, bepridil, raloxifene hydrochloride, benzbromarone,    prazepam, clotiazepam, suloctidil, benzethonium, bicaltamide,    benzthiazide, minaprine, trifluoperazine, chlorprothixene,    pimethixene, flupentixol, clofazimine, loxapine, rescinnamine,    syrosingopine, dihydroergocornine mesylate, dihydro-α-ergocryptine    mesylate, dihydro-β-ergocryptine mesylate, dihydroergocristine    mesylate or stanozolol, or a derivative thereof having the ability    to bind to NCS.-   [36] A substance obtained by the method according to any of the    aforementioned [29]-[35].-   [37] An agent of regulating a pharmacologic action, which comprises    a substance obtained by the method according to any of the    aforementioned [29]-[35].-   [38] A complex, which comprises a drug, and NCS or a mutant protein    thereof.-   [39] A method of producing a complex that comprises a drug and NCS    or a mutant protein thereof, which comprises bringing the drug into    contact with the NCS or the mutant protein.-   [40] A kit, which comprises the following (i) and (ii):-   (i) a drug or a salt thereof;-   (ii) an NCS protein or a mutant protein thereof, nucleic acid that    encodes said protein, expression vector comprising said nucleic acid    or cells that enable a measurement of the expression of the NCS    protein gene.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram showing a SEC interaction screening systemusing a spin column.

FIG. 2 is a schematic diagram showing a SEC interaction analysis using aspin column.

BEST MODE FOR CARRYING OUT THE INVENTION

1. NCS Protein and Gene Thereof

The present invention provides NCS protein and gene.

The NCS protein is a generic term for calcium ion-binding protein whichis specifically expressed in retinal photoreceptor, nerve cell, neuralendocrine cell and the like, and which has EF hand motif. The NCSprotein also has a myristoylation site at its N-terminus. If the NCSprotein bind with Ca²⁺, the localization of the protein to cell membraneis increased by the exposure of myristoyl group arising fromconformational change (see, Biochem. J. 353, 1-12 (2001)).

The examples of the NCS protein include proteins belonging to Frequeninfamily such as NCS-1 (Class A), proteins belonging to VILIPS family suchas neurocalcin α, neurocalcin δ, hypocalcin (Class B), proteinsbelonging to Recoverin family such as Recoverin (Class C), proteinsbelonging to GCAPs family such as GCAP1, GCAP2, or GCAP3 (Class D),proteins belonging to KChIPs family such as KChIP1, KChIP2 or KChIP3(Class E), and the like. The protein is preferably a protein belongingto VILIPS family (Class B), and is more preferably neurocalcin δ andhuman hippocalcin-like protein 1 (or visinin-like protein 3 or VILIP-3).Neurocalcin 6 is known to express in nerve cells such as nerve cells inbrain including peripheral sensory nerve cells and hippocampus dentategyrus involved in memory. The human hippocalcin-like protein 1 (orvisinin-like protein 3 or VILIP-3) is known to localize in foci of braintissue of patient with Alzheimer's disease. As mentioned herein, the NCSprotein is not limited to human NCS proteins, but includes an orthologueof different animal species. The human neurocalcin δ is a proteinderived from FLJ39196 clone described in the Example mentioned below.The human hippocalcin-like protein 1 (or visinin-like protein 3 orVILIP-3) is a protein derived from FLJ20589 clone described in theExample mentioned below.

The NCS protein of the present invention can be, for example, a proteinhaving an amino acid sequence shown by SEQ ID NO:2 or SEQ ID NO:4(VILIP-3). Human NCS protein is registered as FLJ number (registrationnumber in NEDO (New Energy and Industrial Technology Developmentorganization) protein cDNA structural analysis project): FLJ39196,GenBank accession number: AK096515, and H-Inv cDNA ID: HIT000021370 andH-Inv locus ID: HIX0007693 in H-Invitational database (H-Inv DB), aswell as FLJ number: FLJ20589, GenBank accession number: AK000596, andH-Inv cDNA ID: HIT000003071 and H-Inv locus ID: HIX0001817 (see, Nat.Genet. 36(1), 40-45 (2004)), and was found by the present inventors tointeract with the compounds described in the Example, including statindrugs. The NCS protein of the present invention is also registered asOMIM606722 and 600207 in OMIM (Online Mendelian Inheritance in Man™.

According to the present invention, a mutant protein of NCS protein isalso provided. The mutant proteins can be, for example, a protein whichconsists of an amino acid sequence resulting from the substitution,deletion, addition or insertion of one or plural amino acids in theamino acid sequence shown by SEQ ID NO:2 or SEQ ID NO:4 (VILIP-3), andwhich show an interaction with a drug.

The number of amino acids substituted, deleted, added or inserted can beany one that allows the retention of the function, and it can be, forexample about 1 to 30, preferably about 1 to 20, more preferably about 1to 10, further more preferably about 1 to 5, most preferably 1 or 2. Thesite for substitution, deletion, addition or insertion of an amino acidcan be any site that allows the retention of the function, and it canbe, for example, a site of EF hand motif, a myristoylation site andsites other than these sites.

Furthermore, the mutant protein of the present invention can be aprotein which consists of an amino acid sequence having a homology offor example about 50% or more, preferably about 70% or more, morepreferably about 80% or more, further more preferably about 90% or more,most preferably about 95% or more (but excluding 100% homology), to theamino acid sequence shown by SEQ ID NO:2 or SEQ ID NO:4 (VILIP-3), andwhich shows an interaction with a drug. Here, the numerical values ofthe above-described homology are calculated by, for example, executingthe commands for the maximum matching method using the DNASIS sequenceanalytical software (Hitachi Software Engineering). The parameters forthe calculation should be used in default settings (initial settings).

The drug with which the protein of the present invention shows aninteraction is an NCS protein-targeting drug. The NCS protein-targetingdrug refers to a drug which shows a pharmaceutical effect or adverseeffect via NCS protein. The examples of the drug include substancescapable of regulating an action associated with NCS protein-targetingdrug (for example, substances capable of regulating central nervousaction), substances capable of regulating a function associated with NCSprotein gene, and the like. The drug includes a medicine and reagent.Preferably, the NCS protein-targeting drug can be the compound describedbelow.

When the NCS protein of the present invention or a mutant proteinthereof is used, the protein may be labeled or unlabeled. In addition, amixture comprising a labeled protein and an unlabeled protein in aspecified ratio can be used as the protein. The examples of the labelingsubstance include fluorescent substances such as FITC and FAM,luminescent substances such as luminol, luciferin and lucigenin,radioisotopes such as ³H, ¹⁴C, ³²P, ⁵S, and ¹²³I, affinity substancessuch as biotin and streptavidin, and the like.

The NCS protein genes of the present invention may be any ones thatencode the NCS proteins of the present invention. For example, the NCSprotein genes of the present invention can be those corresponding toproteins comprising the above-described amino acid sequences.Preferably, the protein consist of a nucleotide sequence shown by SEQ IDNO:1. The NCS protein genes of the present invention are not limited tohuman genes described above, but include orthologues of different animalspecies.

According to the present invention, the gene is also provided, whichconsists of a nucleotide sequence that hybridizes to a sequencecomplementary to the nucleotide sequence shown by SEQ ID NO:1 understringent conditions, and which corresponds to a protein that shows aninteraction with a drug. Here, hybridize under stringent conditionsmeans that a positive hybridization signal remains observable even underconditions of, for example, heating in a solution of 6×SSC, 0.5% SDS and50% formamide at 42° C., followed by washing in a solution of 0.1×SSCand 0.5% SDS at 68° C.

The NCS proteins of the present invention and genes thereof can be usedfor the development of drugs for diseases associated with NCSprotein-targeting drug (for example, central nervous diseases), ordiseases associated with the NCS protein gene, or for the development ofinvestigational reagents for the diseases, and the like. Hereinafter,each disease is described in detail.

(I. Disease Associated with NCS Protein-Targeting Drug)

“A disease associated with NCS protein-targeting drug” means a diseasefor which NCS protein-targeting drug is used or a disease correspondingto an adverse effect of the drug. The disease associated with NCSprotein-targeting drug can be ameliorated or exacerbated by an NCSprotein-targeting drug. The examples of the disease associated with NCSprotein-targeting drug include central nervous disease and otherdiseases.

“An action associated with NCS protein-targeting drug” means an actionof the same kind as, or opposite kind to, a kind of action actuallyexhibited by an NCS protein-targeting drug (including pharmacologicalactions and adverse effects). In other words, the action associated withNCS protein-targeting drug is an action capable of ameliorate orexacerbate “a disease associated with NCS protein-targeting drug”. Forexample, the action associated with NCS protein-targeting drug iscentral nervous action or anti-central nervous action, if a diseaseassociated with NCS protein-targeting drug is central nervous disease.“An action associated with NCS protein-targeting drug” would be obviousfrom the description of “diseases associated with NCS protein-targetingdrug.”

(Central Nervous Disease)

The central nervous disease is not particularly limited, as long as itinvolves abnormality in central nervous systems, brain and spinal cord.The examples of the central nervous disease include dementia, epilepsy,Parkinson's disease, schizophrenia, anxiety, insomnia, depression, maniaand the like. In particular, dementia is preferred. The dementia can beroughly divided into primary degenerative dementia, and secondarydementia such as multiinfarct dementia (also called cerebrovasculardementia), chronic hydrocephalus, encephalitis, brain tumor,neurosyphilis, pulmonary encephalopathy, drug poisoning or alcoholpoisoning, however, primary degenerative dementia is preferred. Theexamples of primary degenerative dementia include senile dementia whichmainly develops at 65 years old and above, and presenile dementia whichmainly develops at 40-65 years old, such as Alzheimer's disease, Pickdisease or Huntington's disease.

(Other Diseases)

One example of the disease associated with NCS protein-targeting drugcan be a disease associated with atorvastatin.

A diseases associated with atorvastatin means a disease for whichatorvastatin is used or a disease corresponding to an adverse effect ofatorvastatin. Atorvastatin is known as an HMG-CoA reductase inhibitorand the like. Known targets for atorvastatin include Minichromosomemaintenance protein 7, Minichromosome maintenance protein 6, HMG-CoAreductase, and the like. Examples of the disease for which atorvastatinis used include hypercholesterolemia, familial hypercholesterolemia andthe like. In contrast, the examples of the adverse effect ofatorvastatin include gastric discomfort, itching, digital tremor,insomnia, diarrhea, heartburn, constipation, headache, general malaise,rhabdomyolysis, myopathy, impairment of liver function, jaundice,hypersensitivity, thrombocytopenia, skin/mucosa/eye syndrome(Stevens-Johnson syndrome), toxic epidermal necrolysis (Lyell syndrome),erythema multiforme, hyperglycemia, diabetes and the like.

Furthermore, the disease associated with NCS protein-targeting drug canbe a disease associated with NCS-binding compound. Examples of theNCS-binding compounds include pimozide, bifonazole, fendiline,cloperastine, bepridil, raloxifene hydrochloride, benzbromarone,prazepam, clotiazepam, suloctidil, benzethonium, bicaltamide,benzthiazide, minaprine, trifluoperazine, chlorprothixene, pimethixene,flupentixol cis-(Z), clofazimine, loxapine, rescinnamine, syrosingopine,dihydroergotoxine mesylate, dihydroergocristine, stanozolol, andflunarizine.

A disease associated with pimozide means a disease for which pimozide isused or a disease corresponding to an adverse effect of pimozide.Examples of the disease for which pimozide is used includeschizophrenia, abnormal behavior found inmovement/emotion/motivation/interpersonal relation and the likeassociated with autism spectrum disorder or mental retardation in child,pathologic symptom found in sleep/eating/excretion/speech and the like,mental symptom found in stereotypy, and the like. In contrast, examplesof the adverse effect of pimozide include extrapyramidal symptom,parkinson's syndrome (tremor, muscle rigidity, salivation, etc.),akathisia (akathisia), dyskinesia (rotatory nystagmus, dysarthria,dysphagia, etc.), insomnia/drowsiness, manifestation ofagitation/excitation/hyperkinesis/irritability/hallucination/delusion,hypotension, skin rash/itching, nausea/vomiting, anorexia, abdominaldiscomfort, constipation, abdominal pain, diarrhea, dysuria, frequenturination, nocturnal enuresis, increased value of prolactin and thelike.

A disease associated with bifonazole means a disease for whichbifonazole is used or a disease corresponding to an adverse effect ofbifonazole. Examples of the disease for which bifonazole is used includecutaneous fungal disease (trichophytosis, candidiasis, tinea versicolor)and the like. In contrast, examples of the adverse effect of bifonazoleinclude local irritate, dermatitis, redness/erythema, crack, scale,itching, erosion and the like.

A disease associated with cloperastine means a disease for whichcloperastine is used or a disease corresponding to an adverse effect ofcloperastine. Examples of the disease for which cloperastine is usedinclude coughing associated with cold/acute bronchitis/chronicbronchitis/bronchiectasis/pulmonary tuberculosis/lung cancer and thelike. In contrast, examples of the adverse effect of cloperastineinclude drowsiness, nausea, anorexia, dry mouth and the like.

A disease associated with bepridil means a disease for which bepridil isused or a disease corresponding to an adverse effect of bepridil.Examples of the disease for which bepridil is used includetachyarrhythmia, angina and the like. In contrast, examples of theadverse effect of bepridil include QT interval prolongation,bradycardia, ventricular tachycardia, vomiting and the like.

A disease associated with raloxifene hydrochloride means a disease forwhich raloxifene hydrochloride is used or a disease corresponding to anadverse effect of raloxifene hydrochloride. Examples of the disease forwhich raloxifene hydrochloride is used include postmenopausalosteoporosis and the like. In contrast, examples of the adverse effectof raloxifene hydrochloride include thrombocytopenia, decreasedhemoglobin, decreased hematocrit, decreased calcium in blood, decreasedtotal protein in serum, increase of benign intrauterine fluid and thelike.

A disease associated with benzbromarone means a disease for whichbenzbromarone is used or a disease corresponding to an adverse effect ofbenzbromarone. Examples of the disease for which benzbromarone is usedinclude hyperuricemia, gout, hypertension with hyperuricemia and thelike. In contrast, examples of the adverse effect of benzbromaroneinclude abdominal discomfort, gastrointestinal disorder, itching, skinrash, diarrhea, liver disorder and the like.

A disease associated with prazepam means a disease for which prazepam isused or a disease corresponding to an adverse effect of prazepam.Examples of the disease for which prazepam is used includeanxiety/stress/depressive symptom/sleep disorder in neurosis ordepression, physical symptom or anxiety/stress/depressive symptom/sleepdisorder in psychophysiologic disorder (gastrointestinal disease,hypertension, autonomic dystonia) and the like. In contrast, examples ofthe adverse effect of prazepam include drowsiness, dizzy, fatigue,malaise, sluggishness, anorexia and the like.

A disease associated with clotiazepam means a disease for whichclotiazepam is used or a disease corresponding to an adverse effect ofclotiazepam. Examples of the disease for which clotiazepam is usedinclude physical symptom and anxiety/stress/hypochondriasis/depressivesymptom/sleep disorder in psychophysiologic disorder (gastrointestinaldisease, cardiovascular disease), giddiness/neck stiffness/anorexia inautonomic dystonia, and the like. In contrast, examples of the adverseeffect of clotiazepam include drowsiness, dizzy, fatigue, malaise,sluggishness, anorexia and the like.

A disease associated with trifluoperazine means a disease for whichtrifluoperazine is used or a disease corresponding to an adverse effectof trifluoperazine. Examples of the disease for which trifluoperazine isused include schizophrenia and the like. In contrast, examples of theadverse effect of trifluoperazine include akinetic mutism, severe musclerigidity, aphagia, tachycardia, variation in blood pressure, sweating,fall in blood pressure, electrocardiogram abnormality and the like.

A disease associated with clofazimine means a disease for whichclofazimine is used or a disease corresponding to an adverse effect ofclofazimine. Examples of the disease for which clofazimine is usedinclude Hansen's disease and the like. In contrast, examples of theadverse effect of clofazimine include coloring of skin, dry skin,photosensitivity, ichthyosis, skin rash, pruritus, gastrointestinalbleeding, nausea, vomiting, anorexia, giddiness, headache, neuralgia andthe like.

A disease associated with rescinnamine means a disease for whichrescinnamine is used or a disease corresponding to an adverse effect ofrescinnamine. Examples of the disease for which rescinnamine is usedinclude hypertension and the like. In contrast, examples of the adverseeffect of rescinnamine include depression and the like.

A disease associated with dihydroergotoxine mesylate means a disease forwhich dihydroergotoxine mesylate is used or a disease corresponding toan adverse effect of dihydroergotoxine mesylate. Examples of the diseasefor which dihydroergotoxine mesylate is used include hypertension,peripheral circulatory disturbance (Buerger's disease, arteriosclerosisobliterans, arterial embolism, thrombosis, Raynaud's disease andRaynaud's syndrome, acrocyanosis, chilblain/frostbite, intermittentclaudication), metabolic disorder in brain, and the like. In contrast,examples of the adverse effect of dihydroergotoxine mesylate includegastrointestinal disorder, nausea/vomiting, anorexia, skinrash/pruritus, headache, giddiness and the like.

(II. Disease Associated with the NCS Protein Gene)

“A disease associated with the NCS protein gene” refers to a diseasethat can be caused as a result of a change in the function or expressionlevel of the NCS protein gene, or in the function or expression level ofa gene located downstream of the NCS protein gene in the signaltransduction system mediated by the NCS protein gene (downstream gene).The change in the function of the NCS protein gene or a gene downstreamthereof can be caused by, for example, a mutation (e.g., polymorphism)in the gene. Examples of the mutation include a mutation in the codingregion, which promotes or suppresses the function of the gene, amutation in the non-coding region, which promotes or suppresses theexpression thereof, and the like. The change in the expression levelincludes increases or reductions in the expression level. A diseaseassociated with the NCS protein gene can be ameliorated or exacerbatedby NCS protein.

“A function associated with the NCS protein gene” means a function ofthe same kind as, or opposite kind to, the kind of a function that isactually exhibited by NCS protein. In other words, “a functionassociated with the NCS protein gene” is a function capable ofameliorating or exacerbating “a disease associated with the NCS proteingene.” The examples of the function associated with the NCS protein geneinclude phototransduction, regulation of release of bioactive substancessuch as hormone/cytokine and neurotransmitter, control of activity ofguanyl cyclase or adenyl cyclase, regulation of metabolism of cyclicnucleotide, regulation of gene expression, control of ion channel,control of G protein-coupled receptors such as acetylcholine receptorsand dopamine receptors or other cell membrane surface receptors, controlof G protein-coupled receptor kinase, control of proteases such aspresenilin 2, regulation of metabolism of inositol phospholipid, and thelike.

Furthermore, we performed a BLAST search of the NCS protein of thepresent invention in SwissProt and RefSeq, and subjected it to furtheranalysis by GO (Gene Ontology) category classification information onthe basis of the search results. As a result, the protein was classifiedinto GO:0003779¥MF|actin binding, GO:0005509¥MF|calcium ion binding,GO:0015631¥MF|tubulin binding and GO:0030276¥MF|clathrin binding as MF(molecular function); GO:0016192¥BP|vesicle-mediated transport as BP(biological process); and GO:0005829¥CC|cytosol andGO:0030130¥CC|clathrin coat of trans-Golgi network vesicle as CC (cellcomponent). Therefore, the function associated with the NCS protein genecan include functions derivable from these information on GO categoryclassification, in addition to the functions described above.

Since the NCS protein gene is considered to mediate a wide variety ofphysiological functions in the body, a very wide variety of diseases aresupposed as diseases associated with the NCS protein gene. The examplesof the diseases include dementia, epilepsy, Parkinson's disease,schizophrenia, anxiety, insomnia, depression, mania, neurodegeneration,retinal dysplasia, cancer, diabetes, pain (particularly, chronic pain)and the like. Furthermore, other diseases possibly associated with theNCS protein gene include chondrocalcinosis (OMIM600668).

(III. NCS Protein-Targeting Drug)

The (I) to (VIII), the formulas (1) to (11) and the formulas (1′) to(11′) of the present invention are now explained.

Compounds represented by the formulas (I) to (VIII), the formulas (1) to(11) and the formulas (1′) to (11′) can be produced by applying variousknown synthesis methods based on the basic skeleton or the kind ofsubstituent. For example, alkylation, acylation, amination, imination,halogenation, reduction, oxidation, condensation, cyclization and thelike can be mentioned, and the reactions and methods generally used inthis field can be utilized.

The “halogen atom” is a fluorine atom, a chlorine atom, a bromine atomor an iodine atom.

As the “straight chain or branched alkyl having 1 to 9 carbon atoms”,for example, methyl, ethyl, propyl, isopropyl, butyl, isobutyl,sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, sec-pentyl,tert-pentyl, hexyl, isohexyl, 2-ethylbutyl, heptyl, octyl,1,1,3,3-tetramethylbutyl, nonyl and the like can be mentioned.

As the “straight chain or branched alkyl having 1 to 7 carbon atoms”,for example, methyl, ethyl, propyl, isopropyl, butyl, isobutyl,sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, sec-pentyl,tert-pentyl, hexyl, isohexyl, 2-ethylbutyl, heptyl and the like can bementioned.

As the “straight chain or branched alkyl having 1 to 5 carbon atoms”,for example, methyl, ethyl, propyl, isopropyl, butyl, isobutyl,sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, sec-pentyl,tert-pentyl and the like can be mentioned.

The alkyl group is optionally substituted, at any position, by hydroxy,a halogen atom, cyano, methoxy, amino, mono-substituted amino,di-substituted amino, alkylsulfanyl, or halogenated methyl, and to bespecific, hydroxymethyl, 1 or 2-hydroxyethyl, 1, 2 or 3-hydroxypropyland the like can be mentioned.

As the “alkyloxy”, straight chain or branched alkyloxy having 1 to 9carbon atoms, for example, methyloxy, ethyloxy, propyloxy, isopropyloxy,butyloxy, isobutyloxy, sec-butyloxy, tert-butyloxy, pentyloxy,isopentyloxy, neopentyloxy, sec-pentyloxy, tert-pentyloxy, hexyloxy,isohexyloxy, 2-ethylbutyloxy, heptyloxy, octyloxy,1,1,3,3-tetramethylbutyloxy, nonyloxy and the like can be mentioned.

As the “straight chain or branched alkyloxy having 1 to 5 carbon atoms”,for example, methyloxy, ethyloxy, propyloxy, isopropyloxy, butyloxy,isobutyloxy, sec-butyloxy, tert-butyloxy, pentyloxy, isopentyloxy,neopentyloxy, sec-pentyloxy, tert-pentyloxy and the like can bementioned.

As the “straight chain or branched alkyl having 1 to 7 carbon atoms”,for example, methyl, ethyl, propyl, isopropyl, butyl, isobutyl,sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, sec-pentyl,tert-pentyl, hexyl, isohexyl, 2-ethylbutyl, heptyl and the like can bementioned.

As the “phenylalkyl having 7 to 12 carbon atoms”, for example, benzyl, 1or 2-phenylethyl, 1, 2 or 3-phenylpropyl, 1, 2, 3 or 4-phenylbutyl andthe like can be mentioned.

As the “phenylalkyl having 7 to 11 carbon atoms”, for example, benzyl, 1or 2-phenylethyl or 1, 2 or 3-phenylpropyl and the like can bementioned.

As the “phenylalkenyl having 8 to 12 carbon atoms”, for example, 1 or2-phenylethenyl (a or β-styryl), 1, 2 or 3-phenyl-1-propenyl, 1, 2 or3-phenyl-2-propenyl (e.g., cinnamyl etc.), 1, 2, 3 or4-phenyl-1-butenyl, 1, 2, 3 or 4-phenyl-2-butenyl and the like can bementioned.

As the “phenylalkyloxy having 7 to 12 carbon atoms”, for example,benzyloxy, 1 or 2-phenylethyloxy, 1, 2 or 3-phenylpropyloxy, 1, 2, 3 or4-phenylbutyloxy and the like can be mentioned.

As the “alkoxycarbonyloxy”, for example, methoxycarbonyloxy,ethoxycarbonyloxy, propoxycarbonyloxy, isopropoxycarbonyloxy,butoxycarbonyloxy, isobutoxycarbonyloxy, sec-butoxycarbonyloxy,tert-butoxycarbonyloxy and the like can be mentioned.

As the “alkylsulfanyl”, straight chain or branched alkylsulfanyl having1 to 9 carbon atoms, for example, methylsulfanyl, ethylsulfanyl,propylsulfanyl, isopropylsulfanyl, butylsulfanyl, isobutylsulfanyl,sec-butylsulfanyl, tert-butylsulfanyl, pentylsulfanyl,isopentylsulfanyl, neopentylsulfanyl, sec-pentylsulfanyl,tert-pentylsulfanyl, hexylsulfanyl, isohexylsulfanyl,2-ethylbutylsulfanyl, heptylsulfanyl, octylsulfanyl,1,1,3,3-tetramethylbutylsulfanyl, nonylsulfanyl and the like can bementioned.

As the “halogenated alkyl”, straight chain or branched alkyl having 1 to9 carbon atoms and substituted by one or more halogen atoms, forexample, fluoromethyl, difluoromethyl, trifluoromethyl, chloromethyl,dichloromethyl, trichloromethyl, bromomethyl, iodomethyl, 2-chloroethyl,2-fluoroethyl, 2,2,2-trifluoroethyl and the like can be mentioned.

As the “halogenated methyl”, for example, fluoromethyl, difluoromethyl,trifluoromethyl, chloromethyl, dichloromethyl, trichloromethyl,bromomethyl, iodomethyl and the like can be mentioned.

As the “mono-substituted amino”, an amino group mono-substituted by asubstituent selected from the group consisting of the above-definedstraight chain or branched alkyl having 1 to 9 carbon atoms; a phenylgroup optionally having 1 to 3 substituents selected from the groupconsisting of the above-defined straight chain or branched alkyl having1 to 9 carbon atoms, a halogen atom, cyano, hydroxy, methoxy, amino, theabove-defined alkylsulfanyl and the above-mentioned defined halogenatedmethyl; and the like can be mentioned. To be specific, N-methylamino,N-ethylamino, N-propylamino, N-isopropylamino, N-butylamino,N-isobutylamino, N-sec-butylamino, N-tert-butylamino, N-pentylamino,N-isopentylamino, N-neopentylamino, N-sec-pentylamino,N-tert-pentylamino, N-hexylamino, N-isohexylamino, N-2-ethylbutylamino,N-heptylamino, N-octylamino, N-(1,1,3,3-tetramethylbutyl)amino,N-nonylamino, N-phenylamino, N-(2, 3 or 4-chlorophenyl)amino, N-(2, 3 or4-fluorophenyl)amino, N-(2, 3 or 4-methylphenyl)amino, N-(2, 3 or4-methoxyphenyl)amino, N-(2, 3 or 4-methylsulfanylphenyl)amino, N-(2, 3or 4-hydroxyphenyl)amino, N-(2, 3 or 4-cyanophenyl)amino, N-(2, 3 or4-trifluoromethylphenyl)amino and the like can be mentioned.

As the “di-substituted amino”, an amino group di-substituted by the sameor different substituents selected from the group consisting of theabove-defined straight chain or branched alkyl having 1 to 9 carbonatoms; a phenyl group optionally having 1 to 3 substituents selectedfrom the group consisting of the above-defined straight chain orbranched alkyl having 1 to 9 carbon atoms, a halogen atom, cyano,hydroxy, methoxy, amino, the above-defined alkylsulfanyl and theabove-defined halogenated methyl; and the like can be mentioned. To bespecific, N,N-dimethylamino, N,N-diethylamino, N,N-dipropylamino,N,N-diisopropylamino, N,N-dibutylamino, N,N-diisobutylamino,N,N-di-sec-butylamino, N,N-di-tert-butylamino, N,N-dipentylamino,N,N-diisopentylamino, N,N-dineopentylamino, N,N-di-sec-pentylamino,N,N-di-tert-pentylamino, N,N-dihexylamino, N,N-diisohexylamino,N,N-bis(2-ethylbutyl)amino, N,N-diheptylamino, N,N-dioctylamino,N,N-bis(1,1,3,3-tetramethylbutyl)amino, N,N-dinonylamino,N-ethyl-N-methylamino, N-propyl-N-methylamino,N-isopropyl-N-methylamino, N-butyl-N-methylamino,N-isobutyl-N-methylamino, N-sec-butyl-N-methylamino,N-tert-butyl-N-methylamino, N-pentyl-N-methylamino,N-isopentyl-N-methylamino, N-neopentyl-N-methylamino,N-sec-pentyl-N-methylamino, N-tert-pentyl-N-methylamino,N-hexyl-N-methylamino, N-isohexyl-N-methylamino,N-2-ethylbutyl-N-methylamino, N-heptyl-N-methylamino,N-octyl-N-methylamino, N-(1,1,3,3-tetramethylbutyl)-N-methylamino,N-nonyl-N-methylamino, N,N-diphenylamino, N,N-bis(2, 3 or4-chlorophenyl)amino, N,N-bis(2, 3 or 4-fluorophenyl)amino, N,N-bis(2, 3or 4-methylphenyl)amino, N,N-bis(2, 3 or 4-methoxyphenyl)amino,N,N-bis(2, 3 or 4-methylsulfanylphenyl)amino, N,N-bis(2, 3 or4-hydroxyphenyl)amino, N,N-bis(2, 3 or 4-cyanophenyl)amino, N,N-bis(2, 3or 4-trifluoromethylphenyl)amino, N-phenyl-N-methylamino, N-(2, 3 or4-chlorophenyl)-N-methylamino, N-(2, 3 or 4-fluorophenyl)-N-methylamino,N-(2, 3 or 4-methylphenyl)-N-methylamino, N-(2, 3 or4-methoxyphenyl)-N-methylamino, N-(2, 3 or4-methylsulfanylphenyl)-N-methylamino, N-(2, 3 or4-hydroxyphenyl)-N-methylamino, N-(2, 3 or 4-cyanophenyl)-N-methylamino,N-(2, 3 or 4-trifluoromethylphenyl)-N-methylamino and the like can bementioned.

As the “substituted imino”, an imino group substituted by a substituentselected from the group consisting of the above-defined straight chainor branched alkyl having 1 to 9 carbon atoms; a phenyl group optionallyhaving 1 to 3 substituents selected from the group consisting of theabove-defined straight chain or branched alkyl having 1 to 9 carbonatoms, a halogen atom, cyano, hydroxy, methoxy, amino, the above-definedalkylsulfanyl and the above-defined halogenated methyl; and the like canbe mentioned. To be specific, N-methylamino, N-ethylimino,N-propylimino, N-isopropylimino, N-butylimino, N-isobutylimino,N-sec-butylimino, N-tert-butylimino, N-pentylimino, N-isopentylimino,N-neopentylimino, N-sec-pentylimino, N-tert-pentylimino, N-hexylimino,N-isohexylimino, N-2-ethylbutylimino, N-heptylimino, N-octylimino,N-(1,1,3,3-tetramethylbutyl)imino, N-nonylimino, N-phenylimino, N-(2, 3or 4-chlorophenyl)imino, N-(2, 3 or 4-fluorophenyl)imino, N-(2, 3 or4-methylphenyl)imino, N-(2, 3 or 4-methoxyphenyl)imino, N-(2, 3 or4-methylsulfanylphenyl)imino, N-(2, 3 or 4-hydroxyphenyl)imino, N-(2, 3or 4-cyanophenyl)imino, N-(2, 3 or 4-trifluoromethylphenyl)imino and thelike can be mentioned.

As the “cycloalkyl having 3 to 7 carbon atoms”, for example,cyclopropanecyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl andcycloheptyl can be mentioned.

As the “phenylalkyl having 7 to 11 carbon atoms”, for example,phenylmethyl, 1 or 2-phenylethyl, 1, 2 or 3-phenylpropyl, 1, 2, 3 or4-phenylbutyl, 1, 2, 3, 4 or 5-phenylpentyl and the like can bementioned.

As the “imidazolyl”, imidazol-1-yl, imidazol-2-yl, imidazol-4-yl andimidazol-5-yl can be mentioned.

As the “biphenyl”, 2-biphenyl, 3-biphenyl and 4-biphenyl can bementioned.

As the “thienyl”, 2-thienyl and 3-thienyl can be mentioned.

As the “benzothienyl”, 2-benzo[b]thienyl, 3-benzo[b]thienyl,4-benzo[b]thienyl, 5-benzo[b]thienyl, 6-benzo[b]thienyl,7-benzo[b]thienyl, 1-benzo[c]thienyl, 3-benzo[c]thienyl,4-benzo[c]thienyl, 5-benzo[c]thienyl, 6-benzo[c]thienyl and7-benzo[c]thienyl can be mentioned.

As the “benzofuryl”, 2-benzo[b]furyl, 3-benzo[b]furyl, 4-benzo[b]furyl,5-benzo[b]furyl, 6-benzo[b]furyl, 7-benzo[b]furyl, 1-benzo[c]furyl,3-benzo[c]furyl, 4-benzo[c]furyl, 5-benzo[c]furyl, 6-benzo[c]furyl and7-benzo[c]furyl can be mentioned.

As the “piperidinyl”, 1-piperidinyl, 2-piperidinyl, 3-piperidinyl and4-piperidinyl can be mentioned.

As the “pyrrolidinyl”, 1-pyrrolidinyl, 2-pyrrolidinyl and 3-pyrrolidinylcan be mentioned.

As the “piperazinyl”, 1-piperazinyl and 2-piperazinyl can be mentioned.

As the “benzimidazolonyl”, benzimidazol-2-on-1-yl,benzimidazol-2-on-4-yl and benzimidazol-2-on-5-yl can be mentioned.

As the “morpholinyl”, morpholin-1-yl, morpholin-2-yl and morpholin-3-ylcan be mentioned.

As the “phenothiazinyl”, phenothiazin-10-yl, phenothiazin-1-yl,phenothiazin-2-yl, phenothiazin-3-yl and phenothiazin-4-yl can bementioned.

As the “phenazinyl”, phenazinyl-1-yl and phenazinyl-2-yl can bementioned.

As the “dihydrophenazinyl”, 2,10-dihydrophenazinyl-10-yl,2,10-dihydrophenazinyl-10-ylidene and the like can be mentioned.

As the “thioxanthenyl”, thioxanthen-9-yl, thioxanthen-1-yl,thioxanthen-2-yl, thioxanthen-3-yl, thioxanthen-4-yl,thioxanthen-9-ylidene and the like can be mentioned.

As the “dibenzoxazepinyl”, dibenzo[b,f][1,4]oxazepin-11-yl and the likecan be mentioned.

As the “phenoxazinyl”, phenoxazin-10-yl, phenoxazin-1-yl,phenoxazin-2-yl, phenoxazin-3-yl and phenoxazin-4-yl can be mentioned.

As the “acrydinyl”, acridin-9-yl, acridin-1-yl, acridin-2-yl,acridin-3-yl and acridin-4-yl can be mentioned.

As the “xanthenyl”, xanthen-9-yl, xanthen-1-yl, xanthen-2-yl,xanthen-3-yl, xanthen-4-yl, xanthen-9-ylidene and the like can bementioned.

As the “thianthrenyl”, thianthren-1-yl, thianthren-2-yl, thianthren-3-yland thianthren-4-yl can be mentioned.

As the “phenoxathiinyl”, phenoxathiin-1-yl, phenoxathiin-2-yl,phenoxathiin-3-yl and phenoxathiin-4-yl can be mentioned.

As the “divalent pyridazinyl”, pyridazine-3,6-diyl, pyridazine-3,4-diyl,pyridazine-3,5-diyl and pyridazine-3,4-diyl can be mentioned.

As the “straight chain or branched alkylene having 2 to 6 carbon atoms”,for example, ethylene, methylmethylene, trimethylene, methylethylene,ethylmethylene, tetramethylene, 1-methyltrimethylene,2-methyltrimethylene, ethylethylene, propylmethylene,isopropylmethylene, pentamethylene, hexamethylene and the like can bementioned.

As the “straight chain or branched alkylene having 1 to 5 carbon atoms”,for example, methylene, ethylene, methylmethylene, trimethylene,methylethylene, ethylmethylene, tetramethylene, 1-methyltrimethylene,2-methyltrimethylene, ethylethylene, propylmethylene,isopropylmethylene, pentamethylene and the like can be mentioned.

As the “straight chain or branched alkenylene having 2 to 5 carbonatoms”, for example, —CH═CH—, —CH═CH—CH₂—, —C(CH₃)═CH—, —CH═CH—CH₂—CH₂—,—CH₂—CH═CH—CH₂—, —C(CH₃)═CH—CH₂—, —CH═C(CH₃)—CH₂—, —CH═CH—CH(CH₃)—,—C(Et)═CH—, —CH═CH—CH₂—CH₂—CH₂—, —CH₂—CH═CH—CH₂—CH₂—, —CH═CH—CH═CH—,—CH═CH—CH═CH—CH₂— and the like can be mentioned.

As the “straight chain or branched alkynylene having 2 to 5 carbonatoms”, for example, —C≡C—, —C≡C—CH₂—, —C≡C—CH₂—CH₂—, —CH₂—C≡C—CH₂—,—C≡C—CH(CH₃)—, —C≡C—CH₂—CH₂-CH₂—, —CH₂—C≡C—CH₂—CH₂—, —C≡C—CH(CH₃)—CH₂—,—C≡C—CH₂—CH(CH₃)—, —C≡C—CH(Et)— and the like can be mentioned.

As the “straight chain or branched alkylene having 1 to 3 carbon atoms”,for example, methylene, ethylene, methylmethylene, trimethylene,methylethylene and ethylmethylene can be mentioned.

As the “straight chain or branched alkenylene having 2 to 3 carbonatoms”, for example, —CH═CH—, —CH═CH—CH₂— and —C(CH₃)═CH— can bementioned.

As the “alkynylene having 2 to 3 carbon atoms”, for example, —C≡C— and—C≡C—CH₂— can be mentioned.

In the formulas (1) to (VIII), each of R² and R⁴ bonded to each other,R³ and R⁶ bonded to each other, R⁶ and R⁷ bonded to each other, and R⁷and R⁸ bonded to each other may independently forms a ring optionallyhaving 1 to 3 substituents selected from the group consisting of ahalogen atom; cyano; hydroxy; amino; mono-substituted amino;di-substituted amino; halogenated alkyl; alkylsulfanyl;benzimidazolonyl; and straight chain or branched alkyl having 1 to 5carbon atoms or straight chain or branched alkyloxy having 1 to 5 carbonatoms, each of which optionally has 1 to 3 substituents selected fromthe group consisting of a halogen atom, cyano, hydroxy, amino,mono-substituted amino, di-substituted amino, alkyloxy andalkylsulfanyl.

As preferable compounds wherein R² and R⁴, R³ and R⁶, R⁶ and R⁷, and/orR⁷ and R⁸ form a ring, the following compounds can be mentioned.

Compounds wherein R⁷ and R⁸ are bonded to each other to form a ring,which are represented by the following formulas (Ia) to (VIIIa):

wherein each symbol is as defined above;

-   compounds wherein R⁶ and R⁷ are bonded to each other to form a ring,    which are represented by the following formulas (Ib) to (VIIIb):

wherein each symbol is as defined above;

-   compounds wherein R² and R⁴ are bonded to each other to form a ring,    which are represented by the following formulas (Ic) to (VIIc):

wherein each symbol is as defined above;

-   compounds wherein R² and R⁴ are bonded to each other to form a ring,    and R⁶ and R⁷ are bonded to each other to form a ring, which are    represented by the following formulas (Id) to (VIId):

wherein each symbol is as defined above;

-   compounds wherein R³ and R⁶ are bonded to each other to form a ring,    which are represented by the following formulas (Ie) to (VIe) and    (VIIIe):

wherein each symbol is as defined above; and

-   compounds wherein R² and R⁴ are bonded to each other to form a ring,    and R³ and R⁶ are bonded to each other to form a ring, which are    represented by the following formulas (If) to (VIf):

wherein each symbol is as defined above.

As the ring which is formed by R² and R⁴ bonded to each other, forexample, thiochromene, benzothiazine, dihydroquinoxaline, benzopyran,benzoxazine, dihydroquinoline, benzothiazepin, benzoxazepin, benzoxepin,benzothiepin, oxepin, thiepin, oxazepine, thiazepin, thiopyran,dihydropyrazine, pyran, thiazine, oxazine and the like can be mentioned,and it is preferably

and the like, more preferably

and the like.

As the ring which is formed by R³ and R⁶ bonded to each other, forexample, pyrrolidine, piperidine, piperazine, homopiperidine,homopiperazine and the like can be mentioned, with preference given topiperidine and piperazine.

As the ring which is formed by R⁶ and R⁷ bonded to each other, forexample, cyclopentane, cyclohexane, cycloheptane, pyrrolidine,piperidine, piperazine, homopiperidine, homopiperazine, morpholine,thiomorpholine and the like can be mentioned, with preference given topyrrolidine, piperidine and piperazine.

As the ring which is formed by R⁷ and R⁸ bonded to each other, forexample, cyclopentane, cyclohexane, cycloheptane, pyrrolidine,piperidine, piperazine, homopiperidine, homopiperazine, morpholine,thiomorpholine and the like can be mentioned, with preference given topiperidine.

R¹ is preferably a hydrogen atom, a fluorine atom, a chlorine atom,isopropylsulfanyl or 6-hydroxy-2-(4-hydroxyphenyl)benzo[b]thiophen-3-yl.

R² is preferably a hydrogen atom, hydroxy, phenyl, 4-fluorophenyl or2-chlorophenyl, or R² is preferably bonded to R⁴ to form

R³ is preferably a hydrogen atom, methyl or phenyl, or R³ is preferablybonded to R⁶ to form piperidine or piperazine.

R⁴ is preferably a hydrogen atom, or R⁴ is preferably bonded to R² toform

R⁵ is preferably a hydrogen atom or a chlorine atom.

R⁶ is preferably a hydrogen atom, methyl, hexyl, phenyl or cinnamyl, orR⁶ is preferably bonded to R³ to form piperidine or piperazine, orbonded to R⁷ to form pyrrolidine, piperidine, piperazine,1-methylpiperazine, 1-(2-hydroxyethyl)piperazine or4-(benzimidazol-2-on-1-yl)piperidine.

R⁷ is preferably a hydrogen atom, methyl, hexyl, phenyl or cinnamyl, orR⁷ is preferably bonded to R⁶ to form pyrrolidine, piperidine,piperazine, 1-methylpiperazine, 1-(2-hydroxyethyl)piperazine or4-(benzimidazol-2-on-1-yl)piperidine, or bonded to R⁸ to formpiperidine.

R⁸ is preferably a hydrogen atom or isobutyloxymethyl, or R⁸ ispreferably bonded to R⁷ to form piperidine.

As preferable specific examples of the compound represented by theformula (1), Suloctidil can be mentioned.

As preferable specific examples of the compound represented by theformula (II), fendiline can be mentioned.

The formula (III) is preferably the formula (IIIb). As preferablespecific examples of the compound represented by the formula (III),Pimozide can be mentioned.

The formula (IV) is preferably the formula (IVc), the formula (IVd) orthe formula (IVf). As preferable specific examples of the compoundrepresented by the formula (IV), Flupentixol, Chlorprothixene,Pimethixene and the like can be mentioned.

The formula (V) is preferably the formula (Vb), the formula (Ve) or theformula (Vf). As preferable specific examples of the compoundrepresented by the formula (V), Bepridil, Flunarizine, Loxapine and thelike can be mentioned.

The formula (VI) is preferably the formula (VId). As preferable specificexamples of the compound represented by the formula (VI),Trifluoperazine and the like can be mentioned.

The formula (VII) is preferably the formula (VIIb). As preferablespecific examples of the compound represented by the formula (VII),Cloperastine and the like can be mentioned.

The formula (VIII) is preferably the formula (VIIIa). As preferablespecific examples of the compound represented by the formula (VIII),Raloxifene hydrochloride and the like can be mentioned.

As other preferable embodiments of the compounds represented by theformula (1) to the formula (VIII), for example, a compound wherein, inthe formula (1) to the formula (VIII), any one of R¹, R², R³, R⁴, R⁵,R⁶, R⁷ and R⁸ is, instead of the above-mentioned definition, a groupselected from the group consisting of the formulas (B) to (D):

wherein X⁰ is straight chain or branched alkylene having 1 to 5 carbonatoms, and the like can be mentioned.

In the formula (1), Bridge¹ is preferably (1a), R^(1a) is preferably4-fluorophenyl, R^(2a) is preferably phenyl, and R^(3a) is preferablyphenyl.

In the formula (1′), the preferable embodiment is similar to that of theformula (1). In addition, a compound wherein any one of R^(0a)′,R^(4a)′, R^(5a)′ and R^(6a)′ is a group selected from the groupconsisting of the formulas (1B) to (1D):

wherein X^(0a)′ is straight chain or branched alkylene having 1 to 5carbon atoms is also preferable.

In the formula (2), Bridge² is preferably a bridge structure selectedfrom the group consisting of the following formulas

R^(1b) is preferably phenyl, 4-chlorophenyl or 4-fluorophenyl, R^(2b) ispreferably phenyl, 4-fluorophenyl, 4-biphenyl or imidazol-1-yl, andR^(3b) is preferably phenyl, 4-biphenyl, benzimidazol-2-on-1-yl,imidazol-1-yl or piperidin-1-yl.

As preferable specific examples of the compound represented by theformula (2), Pimozide, Bifonazole, Flunarizine, fendiline andCloperastine can be mentioned.

In the formula (2′), the preferable embodiment is similar to that of theformula (2). In addition, a compound wherein any one of R^(4b)′, R^(5b)′and R^(6b)′ is a group selected from the group consisting of theformulas (2B) to (2D):

wherein X^(0b)′ is straight chain or branched alkylene having 1 to 5carbon atoms is also preferable.

In the formula (3), Bridge³ is preferably (3e), R^(1c) is preferablyphenyl, R^(2c) is preferably phenyl, R^(3c) is preferablypyrrolidin-1-yl, and R^(4c) is preferably isobutyl.

As preferable specific examples of the compound represented by theformula (3), Bepridil can be mentioned.

In the formula (3′), the preferable embodiment is similar to that of theformula (3). In addition, a compound wherein any one of R^(0c)′,R^(4c)′, R^(5c)′, R^(6c)′ and R^(7c)′ is a group selected from the groupconsisting of the formulas (3B) to (3D):

wherein X^(0c)′ is straight chain or branched alkylene having 1 to 5carbon atoms is also preferable.

In the formula (4), R^(1d) is preferably 3,5-dibromo-4-hydroxyphenyl or4-[2-(piperidin-1-yl)ethoxy]phenyl, and R^(2d) is preferably2-ethylbenzo[b]furan-3-yl or2-(4-hydroxyphenyl)-6-hydroxybenzo[b]thiophen-3-yl.

As preferable specific examples of the compound represented by theformula (4), Raloxifene hydrochloride and Benzbromarone can bementioned.

In the formula (4′), the preferable embodiment is similar to that of theformula (4). In addition, a compound wherein any one of R^(4d)′ andR^(5d)′ is a group selected from the group consisting of the formulas(4B) to (4D):

wherein X^(0d)′ is straight chain or branched alkylene having 1 to 5carbon atoms is also preferable.

In the formula (5), R^(1e) is preferably phenyl or 2-chlorophenyl,R^(3e) is preferably methyl or isopropylmethyl, and R^(2e) is preferablybenzene or thiophene.

As preferable specific examples of the compound represented by theformula (5), Prazepam and Clotiazepam can be mentioned.

In the formula (5′), the preferable embodiment is similar to that of theformula (5). In addition, a compound wherein any one of R^(3e)′,R^(4e)′, R^(5e)′ and R^(6e)′ is a group selected from the groupconsisting of the formulas (5B) to (5D):

wherein X^(0e)′ is straight chain or branched alkylene having 1 to 5carbon atoms is also preferable.

In the formula (6), Bridge⁶ is preferably a bridge structure selectedfrom the group consisting of the formulas

R^(1f) is preferably phenyl, 4-isopropylphenyl or 4-fluorophenyl, andR^(2f) is preferably phenyl, 4-(1,1,3,3-tetrabutyl)phenyl, octyl,4-cyano-3-trifluoromethylphenyl or a group represented by the formula(f1):

As preferable specific examples of the compound represented by theformula (6), Suloctidil, Benzethonium, Bicaltamide and Benzthiazide canbe mentioned.

In the formula (6′), the preferable embodiment is similar to that of theformula (6). In addition, a compound wherein any one of R^(4f)′ andR^(5f)′ is a group selected from the group consisting of the formulas(6B) to (6D):

wherein X^(0f)′ is straight chain or branched alkylene having 1 to 5carbon atoms is also preferable.

In the formula (7), X¹² is preferably ethylene, R^(1g) is preferablyphenyl, 4-isopropylphenyl or 4-fluorophenyl, R^(2g) is preferablypyridazine-3,6-diyl, and R^(3g) is preferably morpholin-1-yl.

As preferable specific examples of the compound represented by theformula (7), Minaprine can be mentioned.

In the formula (7′), the preferable embodiment is similar to that of theformula (7). In addition, a compound wherein any one of R^(3g)′,R^(4g)′, R^(5g)′ and R^(6g)′ is a group selected from the groupconsisting of the formulas (7B) to (7D):

wherein X^(0g)′ is straight chain or branched alkylene having 1 to 5carbon atoms is also preferable.

In the formulas (8a) to (8j), compounds which are selected from thegroup consisting of the formulas

are preferable. Here, R^(3h) is preferably 4-chlorophenyl,4-methylpiperazin-1-yl, 4-(2-hydroxyethyl)piperazin-1-yl,3-(N,N-dimethylamino)propylidene or 1-methylpiperidin-4-ylidene, andR^(7h) is preferably, thioxanthen-9-ylidene,2-chlorothioxanthen-9-ylidene, 2-trifluoromethylthioxanthen-9-ylidene,2-trifluoromethylphenothiazin-10-yl,2-chlorodibenzo[b,f][1,4]oxazepin-11-yl or a group represented by

As preferable specific examples of the compound represented by theformulas (8a) to (8j), Trifluoperazine, Chlorprothixene, Pimethixene,Flupentixol, Clofazimine and Loxapine can be mentioned.

In the formulas (8a′) to (8j′), the preferable embodiments are similarto those of the formulas (8a) to (8j). In addition, a compound whereinany one of R^(3h)′, R^(5h)′ and R^(6h)′ is a group selected from thegroup consisting of the formulas (8B) to (8D):

wherein X^(0h)′ is straight chain or branched alkylene having 1 to 5carbon atoms is also preferable.

In the formula (9), R^(9i) is preferably 3,4,5-trimethoxyphenyl or3,5-dimethoxy-4-ethoxycarbonyloxyphenyl.

As preferable specific examples of the compound represented by theformula (9), Rescinnamine and Syrosingopine can be mentioned.

In the formula (9′), the preferable embodiment is similar to that of theformula (9). In addition, a compound wherein any one of R^(6i)′ andR^(9i)′ is a group selected from the group consisting of the formulas(9B) to (9D):

wherein X^(0i)′ is straight chain or branched alkylene having 1 to 5carbon atoms is also preferable.

In the formula (10), Bridge¹⁰ is preferably (10a), and R^(11j) ispreferably a group represented by the formula (j2)

wherein X¹⁴ is isopropyl, isobutyl, sec-butyl or benzyl.

As preferable specific examples of the compound represented by theformula (10), Dihydroergocornine mesylate, Dihydro-α-ergocryptinemesylate, Dihydro-β-ergocryptine mesylate and Dihydroergocristinemesylate can be mentioned.

In the formula (10′), the preferable embodiment is similar to that ofthe formula (10). In addition, a compound wherein any one of R^(6j)′ andR^(11j)′ is a group selected from the group consisting of the formulas(10B) to (10D):

wherein X^(0j)′ is straight chain or branched alkylene having 1 to 5carbon atoms is also preferable.

In the formula (11), R^(12k) is preferably (11a), and R^(13k) ispreferably methyl.

As preferable specific examples of the compound represented by theformula (11), Stanozolol can be mentioned.

In the formula (11′), the preferable embodiment is similar to that ofthe formula (11). In addition, a compound wherein any one of R^(6k)′ andR^(13k)′ is a group selected from the group consisting of the formulas(11B) to (11D):

wherein X^(0k)′ is straight chain or branched alkylene having 1 to 5carbon atoms is also preferable.

Any compound represented by the formulas (1) to (VIII), the formulas (1)to (11) and the formulas (1′) to (11′) may be generally referred to asthe compound of the present invention.

Any compound represented by the formulas (1′) to (11′) has an ability tobind to neurocalcin, and can bind to HMG-CoA reductase. Accordingly, itis expected that the compound can exert an action of loweringcholesterol in blood, which is based on HMG-CoA reductase inhibitingactivity, and therefore show extensive effects such as prophylaxis ofcerebrovascular disorder (cerebral infarction, transient cerebralischemia) or dementia due to hyperlipidemia.

The compound of the present invention may form pharmaceuticallyacceptable carrier. The Examples of the salt include acid addition saltssuch as salts with inorganic acids (e.g., hydrochloride, sulfate,hydrobromate, phosphate, etc.) or salts with organic acids (e.g.,acetate, trifluoroacetate, succinate, maleate, fumarate, propionate,citrate, tartrate, lactate, oxalate, methanesulfonate,p-toluenesulfonate, etc.).

The compound of the present invention or a salt thereof may be solvatesuch as hydrate.

2. Screening Method and Product Obtained by the Method

The present invention provides screening methods for drugs, comprisingdetermining whether or not a test substance is capable of regulating theexpression or function of the NCS protein gene. The screening methods ofthe present invention can be roughly divided into a screening method forsubstances capable of regulating an action associated with NCSprotein-targeting drug (for example, central nervous action), and ascreening method for substances capable of regulating a functionassociated with the NCS protein gene, from the viewpoint of the kind ofdrug to be screened. The screening methods of the present invention canalso be performed in vitro, in vivo or in silico. The substance capableof regulating the expression of NCS protein gene, which is obtained bythe screening method of the present invention, has the same definitionas a substance capable of regulating the amount of NCS protein, and canbe a substance capable of altering an amount of NCS protein existed in acertain tissue or cell, or an amount of NCS protein existed in a certainsubcellular localization. Therefore, the examples of the substancecapable of regulating the expression of NCS protein gene include notonly a substance capable of regulating biosynthesis of NCS protein fromNCS protein gene, but also a substance capable of regulating subcellularlocalization of NCS protein and a substance capable of regulatingmetabolism (e.g., degradation associated with metabolism) of NCSprotein. The individual screening methods are hereinafter described indetail.

2.1. Screening Method for Substances Capable of Regulating an ActionAssociated with NCS Protein-Targeting Drug (Screening Method I)

The present invention provides a screening method for substances capableof regulating an action associated with NCS protein-targeting drug,which comprises determining whether or not a test substance is capableof regulating the expression or function of the NCS protein gene.

This screening method is optionally abbreviated as “screening method I.”

Screening method I can be roughly divided into two types: a screeningmethod for a substance capable of regulating an action associated withNCS protein-targeting drug, which comprises determining whether or not atest substance is capable of regulating the expression or function ofNCS protein gene, and selecting a test substance capable of regulatingthe expression or function of NCS protein gene (screening method Ia),and a screening method for a substance capable of regulating an actionassociated with NCS protein-targeting drug (particularly an actionassociated with known target molecule), which comprises determiningwhether or not a test substance is capable of regulating the expressionor function of NCS protein gene, and selecting a test substance that isincapable of regulating the expression or function of NCS protein gene(screening method Ib). Screening method Ia can be useful for thedevelopment of regulators of diseases or conditions associated with NCSprotein-targeting drug and the like. Screening method Ib can be usefulfor the development of drugs capable of regulating an action associatedwith known target molecule, and showing decreased adverse effects of NCSprotein-targeting drug and the like.

2.1.1. Screening Method for Substances Capable of Regulating an ActionAssociated with NCS Protein-Targeting Drug, which Comprises Selecting aTest Substance Capable of Regulating the Expression or Function of NCSProtein Gene (Screening Method Ia)

The test substance subjected to the screening method may be any knowncompound or new compound; examples include nucleic acids, saccharides,lipids, proteins, peptides, organic small compounds, compound librariesprepared using combinatorial chemistry technique, random peptidelibraries prepared by solid phase synthesis or the phage display method,or natural components derived from microorganisms, animals, plants,marine organisms and the like, and the like.

The test substance may be labeled or unlabeled. In addition, a mixturecomprising a labeled substance and an unlabeled substance in a specifiedratio can be used as the test substance. The labeling substance is thesame as described above.

In one embodiment, screening method Ia comprises the following steps(a), (b) and (c):

-   (a) a step for bringing the test substance into contact with NCS    protein;-   (b) a step for measuring the functional level of the protein in the    presence of the test substance, and comparing this functional level    with the functional level of the protein in the absence of the test    substance;-   (c) a step for selecting a test substance that alters the functional    level of the protein on the basis of the result of the comparison in    step (b) above.

The methodology comprising the above-described steps (a) to (c) isreferred to as “methodology I” as required.

In step (a) of methodology I, a test substance is brought into contactwith NCS protein. Contact of the test substance with the protein can beperformed by contact of isolated NCS protein and the test substance insolution, or contact of cells or tissue capable of expressing NCSprotein, and the test substance.

NCS protein can be prepared by a method known per se. For example, NCSprotein can be isolated and purified from the above-described expressiontissue. However, to prepare NCS protein quickly, easily, and in largeamounts, and to prepare human NCS protein, it is preferable to prepare arecombinant protein by gene recombination technology. The recombinantprotein may be prepared using a cell system or a cell-free system.

The cells capable of expressing NCS protein can be any cells thatexpress NCS protein; examples include cells derived from the tissue inwhich NCS protein is expressed, cells transformed with NCS proteinexpression vector and the like. Those skilled in the art are able toeasily identify or prepare these cells; useful cells include primaryculture cells, cell lines derivatively prepared from the primary culturecells, commercially available cell lines, cell lines available from cellbanks, and the like. As the tissue capable of expressing NCS protein,the above-described expression tissues can be used.

In step (b) of methodology I, the functional level of the protein in thepresence of the test substance is measured. A measurement of thefunctional level can be performed according to the method known per sewhich can measure the function of NCS protein.

The functional level may also be measured on the basis of the functionallevel of NCS protein to each isoform (e.g., splicing variant) or theisoform-isoform functional level ratio, rather than on the basis of thetotal functional level of NCS protein.

Next, the functional level of NCS protein in the presence of the testsubstance is compared with the functional level of NCS protein in theabsence of the test substance. This comparison of functional level ispreferably performed on the basis of the presence or absence of asignificant difference. Although the functional level of NCS protein inthe absence of the test substance may be measured prior to, orsimultaneously with, the measurement of the functional level of NCSprotein in the presence of the test substance, it is preferable, fromthe viewpoint of experimental accuracy and reproducibility, that thefunctional level be measured simultaneously.

In step (c) of methodology I, a test substance that alters thefunctional level of the protein is selected. The test substance thatalters the functional level of the protein is capable of promoting orsuppressing a function of NCS protein. The test substance thus selectedcan be useful for the regulation of a disease or condition associatedwith NCS protein-targeting drug.

In another embodiment, screening method Ia comprises the following steps(a), (b) and (c):

-   (a) a step for bringing a test substance into contact with cells    enabling a measurement of the expression of the NCS protein gene;-   (b) a step for measuring the expression level in the cells in    contact with the test substance, and comparing this expression level    with the expression level in control cells not in contact with the    test substance;-   (c) a step for selecting a test substance that regulates the    expression level of the NCS protein gene on the basis of the result    of the comparison in step (b) above.

The methodology comprising the above-described steps (a) to (c) isoptionally abbreviated as “methodology II.”

In step (a) of methodology II, a test substance is brought into contactwith cells enabling a measurement of the expression of the NCS proteingene. Contacting the test substance with the cells enabling ameasurement of the expression of the NCS protein gene can be performedin culture medium.

“Cells enabling a measurement of the expression of the NCS protein gene”refers to cells enabling a direct or indirect evaluation of theexpression level of a product, for example, a transcription product ortranslation product, of the NCS protein gene. The cells enabling adirect evaluation of the expression level of a product of the NCSprotein gene can be cells capable of naturally expressing the NCSprotein gene, whereas the cells enabling an indirect evaluation of theexpression level of a product of the NCS protein gene can be cellsenabling a reporter assay on the transcription regulatory region for theNCS protein gene.

The cells capable of naturally expressing the NCS protein gene can beany cells that potentially express the NCS protein gene; examplesinclude cells showing permanent expression of the NCS protein gene,cells that express the NCS protein gene under inductive conditions(e.g., drug treatment) and the like. Those skilled in the art are ableto easily identify these cells; useful cells include primary culturecells, cell lines derivatively prepared from the primary culture cells,commercially available cell lines, cell lines available from cell banks,and the like. The cell lines expressing neurocalcin δ of NCS proteinsinclude optic nerve cells, peripheral sensory nerve cells, nerve cellsin brain, or lines derived from these cells.

The cells enabling a reporter assay on the transcription regulatoryregion for the NCS protein gene are cells incorporating thetranscription regulatory region for the NCS protein gene and a reportergene functionally linked to the region. The transcription regulatoryregion for the NCS protein gene and the reporter gene are inserted in anexpression vector.

The transcription regulatory region for the NCS protein gene may be anyregion enabling the control of the expression of the NCS protein gene;examples include a region from the transcription initiation point toabout 2 kbp upstream thereof, and a region consisting of a nucleotidesequence wherein one or more nucleotides are deleted, substituted oradded in the nucleotide sequence of the region, and that is capable ofcontrolling the transcription of the NCS protein gene, and the like.

The reporter gene may be any gene that encodes a detectable protein orenzyme; examples include the GFP (green fluorescent protein) gene, GUS(β-glucuronidase) gene, LUS (luciferase) gene, CAT (chloramphenicolacetyltransferase) gene and the like.

The cells transfected with the transcription regulatory region for theNCS protein gene and the reporter gene functionally linked to the regionare not subject to limitation, as long as they enable an evaluation ofthe transcription regulatory function of the NCS protein gene, that is,as long as they enable a quantitative analysis of the expression levelof the reporter gene. However, the cells transfected are preferablycells capable of naturally expressing the NCS protein gene because theyare considered to express a physiological transcription regulatoryfactor for the NCS protein gene, and therefore to be more appropriatefor the evaluation of the regulation of the expression of the NCSprotein gene.

The culture medium in which a test substance and cells enabling ameasurement of the expression of the NCS protein gene are brought intocontact with each other is chosen as appropriate according to the kindof cells used and the like; examples include minimal essential medium(MEM) containing about 5 to 20% fetal bovine serum, Dulbecco's modifiedminimal essential medium (DMEM), RPMI1640 medium, 199 medium and thelike. Culture conditions are also determined as appropriate according tothe kind of cells used and the like; for example, the pH of the mediumis about 6 to about 8, culture temperature is normally about 30 to about40° C., and culture time is about 12 to about 72 hours.

In step (b) of methodology II, first, the expression level of the NCSprotein gene in the cells in contact with the test substance ismeasured. The measurement of expression level can be performed by amethod known per se in view of the kind of cells used and the like.

For example, when cells capable of naturally expressing the NCS proteingene are used as the cells enabling a measurement of the expression ofthe NCS protein gene, the expression level can be measured by a methodknown per se with a product, for example, a transcription product ortranslation product, of the NCS protein gene as the subject. Forexample, the expression level of a transcription product can be measuredby preparing total RNA from the cells, and performing RT-PCR, Northernblotting and the like. The expression level of a translation product canbe measured by preparing an extract from the cells, and performing animmunological technique. Useful immunological techniques includeradioisotope immunoassay (RIA), ELISA (Methods in Enzymol. 70: 419-439(1980)), fluorescent antibody method and the like.

On the other hand, when cells enabling a reporter assay on thetranscription regulatory region for the NCS protein gene are used as thecells enabling a measurement of the expression of the NCS protein gene,the expression level can be measured on the basis of the signalintensity of the reporter.

The expression level may also be measured on the basis of the expressionlevel of NCS protein gene to each isoform (e.g., splicing variant) orthe isoform-isoform expression ratio, rather than on the basis of thetotal functional level of NCS protein gene.

Furthermore, the expression level can be measured based on localizationto cell membrane. The amount of NCS protein localized in a cell can bemeasured by a method known per se. For example, NCS protein fused with agene encoding a fluorescent protein such as GFP gene is introduced to asuitable cell, and the cell is cultured in a culture medium in thepresence of a test substance. Next, fluorescent signal in the cellmembrane is observed by confocal microscopy, and the fluorescent signalmay be compared with fluorescent signal in the organ in the absence ofthe test substance. Furthermore, the amount of NCS protein localized tocell membrane can also be measured by immunostaining using an antibodyagainst NCS protein.

Next, the expression level of the NCS protein gene in the cells incontact with the test substance is compared with the expression level ofthe NCS protein gene in control cells not in contact with the testsubstance. This comparison of expression level is preferably performedon the basis of the presence or absence of a significant difference.Although the expression level of the NCS protein gene in the controlcells not in contact with the test substance may be measured prior to,or simultaneously with, the measurement of the expression level of theNCS protein gene in the cells in contact with the test substance, it ispreferable, from the viewpoint of experimental accuracy andreproducibility, that the expression level be measured simultaneously.

In step (c) of methodology II, a test substance that regulates theexpression level of the NCS protein gene is selected. The regulation ofthe expression level of the NCS protein gene can be the promotion orsuppression of the expression level. The test substance thus selectedcan be useful for the regulation of an action associated with NCSprotein-targeting drug.

In another embodiment, screening method Ia comprises the following steps(a), (b) and (c):

-   (a) a step for bringing a test substance into contact with NCS    protein or a mutant protein thereof;-   (b) a step for measuring the ability of the test substance to bind    to the protein;-   (c) a step for selecting a test substance having the ability of    binding to the protein on the basis of the results of step (b)    above.

The methodology comprising the above-described steps (a) to (c) isoptionally abbreviated as “methodology III.”

In step (a) of methodology III, a test substance is brought into contactwith NCS protein. Contacting the test substance with the protein can beperformed by mixing the test substance and the protein in solution.

NCS protein can be prepared by a method known per se. For example, NCSprotein can be isolated and purified from the above-described expressiontissue of the NCS protein gene. However, to prepare NCS protein quickly,easily, and in large amounts, and to prepare human NCS protein, it ispreferable to prepare a recombinant protein by gene recombinationtechnology. The recombinant protein may be prepared using a cell systemor a cell-free system. The mutant protein having the ability of bindingto an NCS protein-targeting drug also can be easily prepared by thoseskilled in the art with a method known per se. The mutant protein is asdescribed above.

In step (b) of methodology III, the ability of the test substance tobind to the protein is measured. The measurement of the binding abilitycan be performed by a method known per se. Furthermore, the bindingstrength, the dose dependency of the test substance in binding to theprotein, and the like can also be measured, in addition to the bindingability. The binding strength and the dose dependency can be measured byappropriately selecting a measuring means.

The measurement of the binding ability can be performed by, for example,the SEC/MS (size exclusion chromatography/mass analysis) method (seeMoy, F. J. et al., Anal. Chem., 2001, 73, 571-581). The SEC/MS methodcomprises (1) a step for adding a mixed multiplied compound standard tothe purified protein, and then separating the free compound and theprotein by SEC, and (2) an analytical step for identifying the boundcompound contained in the protein fraction by MS. The SEC/MS method isadvantageous in that the binding ability can be analyzed while both theprotein and the test substance are in non-modified and non-immobilizedstate. In the SEC/MS method, not only the ability of the test substanceto bind to the protein, but also the dose dependency of the testsubstance in the binding to the protein and the like can be measuredsimultaneously.

The measurement of the binding ability can also be performed using ameans for measurement based on surface plasmon resonance, for example,Biacore. Using Biacore, the binding and dissociation of a test substanceto a protein immobilized on a chip are measured, and the measured valuesare compared with those obtained when a solution not containing the testsubstance is loaded on the chip. Subsequently, a test substance capableof binding to the protein is selected on the basis of the result for thebinding and dissociation rate or binding amount. Biacore also enablessimultaneous measurements of binding strength (e.g., K_(d) value) andthe like, in addition to the ability of a test substance to bind to aprotein.

Other methods for measuring the binding ability include, for example,SPR-based methods or optical methods such as the quartz crystalmicrobalance (QCM) method, the dual polarization interferometer (DPI)method, and the coupled waveguide plasmon resonance method,immunoprecipitation, isothermal titration and differential scanningcalorimetry, capillary electrophoresis, energy transfer, fluorescentanalytical methods such as fluorescent correlation analysis, andstructural analytical methods such as X-ray crystallography and nuclearmagnetic resonance (NMR).

In measuring the binding ability, an NCS protein-binding substance canalso be used as a control.

“A NCS protein-binding substance” is a compound capable of interactingdirectly with NCS protein or a mutant protein thereof, and can be, forexample, a protein, a nucleic acid, a carbohydrate, a lipid, or a smallorganic compound. Preferably, the NCS protein-binding substance can bethe NCS protein-targeting drug described above, and can be, for example,atorvastatin, pimozide, bifonazole, flunarizine, fendiline,chloperastine, bepridil, raloxifene hydrochloride, benzbromarone,prazepam, clotiazepam, suloctidil, benzethonium, bicaltamide,benzthiazide, minaprine, trifluoperazine, chlorprothixene, pimethixene,flupentixol, clofazimine, loxapine, rescinnamine, syrosingopine,dihydroergocornine mesylate, dihydro-α-ergocryptine mesylate,dihydro-β-ergocryptine mesylate, dihydroergocristine mesylate orstanozolol, or a derivative thereof capable of binding to NCS protein,or a salt thereof.

Although the salts may be any salts, pharmaceutically acceptable saltsare preferable; examples include salts with inorganic bases (e.g.,alkali metals such as sodium and potassium; alkaline earth metals suchas calcium and magnesium; aluminum, ammonium), salts with organic bases(e.g., trimethylamine, triethylamine, pyridine, picoline, ethanolamine,diethanolamine, triethanolamine, dicyclohexylamine,N,N-dibenzylethylenediamine), salts with inorganic acids (e.g.,hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid,phosphoric acid), salt with organic acids (e.g., formic acid, aceticacid, trifluoroacetic acid, fumaric acid, oxalic acid, tartaric acid,maleic acid, citric acid, succinic acid, malic acid, methanesulfonicacid, benzenesulfonic acid, p-toluenesulfonic acid), salts with basicamino acids (e.g., arginine, lysine, ornithine) or salts with acidicamino acids (e.g., aspartic acid, glutamic acid) and the like.

Furthermore, the binding ability may also be measured on the basis ofthe binding ability of NCS protein to each isoform (e.g., splicingvariant) or the isoform-isoform binding ability ratio, rather than onthe basis of the total binding ability of NCS protein.

The binding ability can also be measured in silico. For example, ameasurement of the binding ability can be performed on the basis of SBDD(structure-based drug design: SBDD) or CADD (computer-aided drugdesign). Examples of such screening include virtual screening, de novodesign, pharmacophore analysis, QSAR (quantitative structure activityrelationship) and the like. If information on the steric structure ofthe protein or the target site of the protein is required during suchscreening, the information on the steric structure is used, providedthat the steric structure is known by a structural analytical techniquesuch as NMR, X-ray crystallographic analysis, or synchrotron radiationanalysis. If the steric structure is unknown, information obtained by astructural estimation method such as the homology method or thethreading method is used. In virtual screening, a program known per secan be used; examples of the program include DOCK (Kuntz, I. D. et al.,Science, 1992, 257, 1078), Gold (Jones, G. et al., J. Mol. Biol., 1995,245, 43), FlexX (Rarey, M. et al., J. Mol. Biol., 1996, 261, 470),AutoDock (Morris, G. M. et al., J. Comput. Chem., 1998, 19, 1639), ICM(Abagyan, R. A. et al., J. Comput. Chem., 1994, 15, 488) and the like.

In step (c) of methodology III, a test substance capable of binding toNCS protein is selected. The test substance capable of binding to theprotein is capable of promoting or suppressing a function of the NCSprotein gene. The test substance thus selected can be useful for theregulation of a disease associated with NCS protein-targeting drug.

In still another embodiment, screening method Ia comprises the followingsteps (a), (b) and (c):

-   (a) a step for bringing a test substance and an NCS protein-binding    substance into contact with NCS protein or a mutant protein thereof;-   (b) a step for measuring the amount of the NCS protein-binding    substance to bind to the protein in the presence of the test    substance, and comparing this binding amount with the amount of the    NCS protein-binding substance to bind to the protein in the absence    of the test substance;-   (c) a step for selecting a test substance that alters the amount of    the NCS protein-binding substance to bind to the protein on the    basis of the result of the comparison in step (b) above.

The methodology comprising the above-described steps (a) to (c) isoptionally abbreviated as “methodology IV.”

In step (a) of methodology IV, both a test substance and the NCSprotein-binding substance are brought into contact with NCS protein or amutant protein thereof. Contacting the test substance and the NCSprotein-binding substance with the protein can be performed by mixingthe test substance, the NCS protein-binding substance, and the proteinin solution. The order of bringing the test substance and NCSprotein-binding substance into contact with the protein is not subjectto limitation; one of them may be brought into contact with the proteinin advance or at the same time.

NCS protein and the mutant protein thereof can be prepared by a methodknown per se. For example, preparation of the protein can be performedby a method described in methodology III above.

The NCS protein-binding substance may be labeled or unlabeled. Inaddition, a mixture comprising a labeled substance and an unlabeledsubstance in a specified ratio can be used as the NCS protein-bindingsubstance. The labeling substance is the same as described above.

In step (b) of methodology IV, first, the amount of the NCSprotein-binding substance to bind to the protein is measured in thepresence of the test substance. The measurement of the binding amountcan be performed by a method known per se, in view of kinds of the NCSprotein-binding substance used, the presence or absence of the label,and the like. Furthermore, the binding strength (for example, K_(d)value), the dose dependency of the test substance in the binding to theprotein, and the like can also be measured, in addition to the bindingamount. The binding strength and the dose dependency can be measured byappropriately selecting a measuring means.

The measurement of the binding amount can be performed using, forexample, a labeled NCS protein-binding substance. The NCSprotein-binding substance bound to the protein and the unbound NCSprotein-binding substance may be separated before measuring the bindingamount. More specifically, the measurement can be performed in the samemanner as methodology III.

The binding ability may also be measured on the basis of the bindingability of NCS protein to each isoform (e.g., splicing variant) or theisoform-isoform binding ability ratio, rather than on the basis of thetotal amount of NCS protein bound.

Next, the binding amount of the NCS protein-binding substance to theprotein in the presence of the test substance is compared with thebinding amount of the NCS protein-binding substance to the protein inthe absence of the test substance. The comparison of the binding amountis preferably performed on the presence or absence of a significantdifference. Although the binding amount of the NCS protein-bindingsubstance to the protein in the absence of the test substance may bemeasured prior to, or simultaneously with, the measurement of thebinding amount of the NCS protein-binding substance to the protein inthe presence of the test substance, it is preferable, from the viewpointof experimental accuracy and reproducibility, that the binding amount bemeasured simultaneously.

In step (c) of methodology IV, a test substance that alters the amountof the NCS protein-binding substance to bind to the protein is selected.The change in the binding amount can be, for example, a reduction orincrease of binding amount, with preference given to a reduction ofbinding amount. The test substance thus selected can be useful for theregulation of an action associated with NCS protein-targeting drug.

In addition, screening method Ia can further comprise (d) (i) a step forconfirming that the selected test substance is capable of regulating,for example, promoting or suppressing, an action associated with NCSprotein-targeting drug (confirmation step), or (ii) a step foridentifying the kind of action exhibited by the selected test substance(identification step). The confirmation step or identification step canbe performed by, for example, administering the test substance to anormal animal, or to an animal with “a disease associated with NCSprotein-targeting drug” or model animal. Alternatively, these steps canalso be performed by contacting a test substance with cells, andevaluating change in phenotypes of the contacted cells. According tothis identification step, the kind of “action associated with NCSprotein-targeting drug” exhibited by the selected test substance can bedetermined, and whether or not the selected test substance can be usedas either a drug or an investigational reagent, or both a drug or aninvestigational reagent, and the kind of drug or investigational reagentto which the test substance is applicable can be confirmed.

Screening method Ia can also be performed by administration of a testsubstance to an animal. In this case, not only the expression level ofNCS protein gene, but also the expression level of NCS protein (e.g.,the amount of NCS protein existed in a certain tissue or cell from ananimal administered with a test substance, or localization in cellmembrane) may be measured. Examples of the animal include mammals suchas mice, rats, hamsters, guinea pigs, rabbits, dogs, and monkeys, andbirds such as chickens. When a screening method of the present inventionis performed using an animal, for example, a test substance thatregulates the expression level of the NCS protein gene can be selected.

Screening method Ia enables screening of a substance capable ofregulating an action associated with NCS protein-targeting drug. Hence,screening method Ia is useful for the development of a prophylactic ortherapeutic agent for a disease associated with NCS protein-targetingdrug (for example, central nervous disease), and an investigationalreagent for the disease, and the like.

2.1.2. Screening Method for Substances Capable of Regulating an ActionAssociated with NCS Protein-Targeting Drug, which Comprises Selecting aTest Substance Incapable of Regulating the Expression or Function of NCSProtein Gene (Screening Method Ib)

The present invention provides a screening method for substances capableof regulating an action associated with NCS protein-targeting drug(particularly an action associated with known target molecule and/or apharmacological action actually shown by NCS protein-targeting drug)(for example, a substance which can be used for the same medical use asNCS protein-targeting drug, which exerts a pharmacological actionactually shown by NCS protein-targeting drug, and does not exert adverseeffect actually shown by NCS protein-targeting drug or exert decreasedadverse effect), which comprises determining whether or not a testsubstance is capable of regulating the expression or function of NCSprotein gene, and selecting a test substance incapable of regulating theexpression or function of NCS protein gene.

Screening method Ib can be performed in the same manner as methodologiesI to IV except that a test substance that does not cause a change ordoes not have the binding ability or regulatory capacity in step (c) ofthe above-described methodologies I to IV is selected.

In screening method Ib, the test substance used can be one capable ofregulating the expression or function of a known target molecule, or onehaving an action associated with NCS protein-targeting drug(particularly, a pharmacological action actually showed by NCSprotein-targeting drug). Hence, screening method Ib can be used incombination with a screening method for substances capable of regulatingan action associated with a known target molecule, which comprisesdetermining whether or not the test substance is capable of regulatingthe expression or function of the known target molecule. The screeningmethod for substances capable of regulating an action associated with aknown target molecule can be performed in the same manner as theabove-described screening method Ia. Alternatively, screening method Ibcan be used in combination with a screening method for substancescapable of regulating an action associated with NCS protein-targetingdrug, which comprises determining whether or not the test substance iscapable of regulating the action associated with NCS protein-targetingdrug (particularly a pharmaceutical action actually shown by NCSprotein-targeting drug). Such a screening method can be performed usingan animal or cells in the same manner as the step (d) of theabove-described screening method Ia.

Screening method Ib enables the development of drugs capable ofregulating an action associated with a known target molecule, andshowing decreased adverse effects of NCS protein-targeting drug. Hence,screening method Ib is useful for the improvement of existing drugscapable of regulating an action associated with a known target molecule,and the like.

2.2. Screening Method for Substances Capable of Regulating a FunctionAssociated with the NCS Protein Gene (Screening Method II)

The present invention provides a screening method for substances capableof regulating a function associated with the NCS protein gene, whichcomprises evaluating whether or not a test substance is capable ofregulating the ability of an NCS protein-targeting drug to bind to NCSprotein or a mutant protein thereof.

This screening method is optionally abbreviated as “screening methodII.”

In one embodiment, screening method II comprises the following steps(a), (b) and (c):

-   (a) a step for bringing a test substance and an NCS    protein-targeting drug into contact with NCS protein or a mutant    protein thereof capable of binding to the NCS protein-targeting    drug;-   (b) a step for measuring the amount of the NCS protein-targeting    drug to bind to the protein in the presence of the test substance,    and comparing this binding amount with the amount of the NCS    protein-targeting drug to bind to the protein in the absence of the    test substance;-   (c) a step for selecting a test substance that alters the amount of    the NCS protein-targeting drug to bind to the protein on the basis    of the result of the comparison in step (b) above.

The methodology comprising the above-described steps (a) to (c) is thesame as methodology IV except that “an NCS protein-targeting drug” isused instead of “an NCS protein-binding substance.”

Screening method II enables, for example, screening of substancescapable of regulating a function associated with the NCS protein gene,probes for NCS protein, and the like. Hence, screening method II isuseful for the screening of prophylactic or therapeutic agents fordiseases associated with the NCS protein gene, and investigationalreagents for the diseases, and the like.

2.3. Products Obtained by Screening Methods

The present invention provides products obtained by the above-describedscreening methods, for example, screening methods I and II.

The product provided by a screening method of the present invention canbe a substance obtained by the screening method of the presentinvention, or a regulator of pharmacological action, comprising asubstance obtained by the screening method.

The product provided by the screening method of the present invention isuseful for, for example, the prevention or treatment of a diseaseassociated with NCS protein-targeting drug, or a disease associated withthe NCS protein gene, or as an investigational reagent for the disease,or the like.

3. Regulator

The present invention provides regulators of pharmacological action,each of which a substance that regulates the expression or function ofthe NCS protein gene. The regulators of the present invention can beroughly divided into a regulator of an action associated with NCSprotein-targeting drug (e.g., central nervous action), and a regulatorof a function associated with the NCS protein gene, from the viewpointof the pharmacological action to be regulated. The individual regulatorsare hereinafter described in detail.

3.1. Regulator of an Action Associated with NCS Protein-Targeting Drug(Regulator I)

The present invention provides a regulator of an action associated withNCS protein-targeting drug, comprising a substance that regulates theexpression or function of the NCS protein gene.

This regulator is optionally abbreviated as “regulator I.”

The substance that regulates the expression or function of the NCSprotein gene can be, for example, a substance that suppresses theexpression of the NCS protein gene. The expression refers to a state inwhich a translation product of the NCS protein gene is produced and islocalized at the action site thereof in a functional condition. Hence,the substance that suppresses the expression may be one that acts in anystage of gene transcription, post-transcriptional regulation,translation, post-translational modification, localization and proteinfolding and the like.

Specifically, the substance that suppresses the expression of the NCSprotein gene is exemplified by transcription suppressor, RNA polymeraseinhibitor, RNA decomposing enzyme, protein synthesis inhibitor, nucleartranslocation inhibitor, protein decomposing enzyme, protein denaturantand the like; to minimize the adverse effects on other genes andproteins expressed in the cells, it is important that the substance becapable of specifically acting on the target molecule.

An example of the substance that suppresses the expression of the NCSprotein gene is an antisense nucleic acid to a transcription product,specifically, mRNA or initial transcription product, of the NCS proteingene. “An antisense nucleic acid” refers to a nucleic acid that consistsof a nucleotide sequence capable of hybridizing to the target mRNA(initial transcription product) under physiological conditions for cellsthat express target mRNA (initial transcription product), and capable ofinhibiting the translation of the polypeptide encoded by the target mRNA(initial transcription product) in a hybridized state. The kind ofantisense nucleic acid may be DNA or RNA, or a DNA/RNA chimera. Becausea natural type antisense nucleic acid easily undergoes degradation ofthe phosphoric acid diester bond thereof by a nucleic acid decomposingenzyme present in the cells, an antisense nucleic acid of the presentinvention can also be synthesized using a modified nucleotide such asthe thiophosphate type (P═O in phosphate linkage replaced with P═S), and2′-O-methyl type which are stable to decomposing enzymes. Otherimportant factors for the designing of antisense nucleic acid includeincreases in water-solubility and cell membrane permeability and thelike; these can also be cleared by choosing appropriate dosage formssuch as those using liposome or microspheres.

The length of antisense nucleic acid is not subject to limitation, aslong as the antisense nucleic acid is capable of specificallyhybridizing to the transcription product of the NCS protein gene; It maybe of a sequence complementary to a sequence of about 15 bases for theshortest, or the entire sequence of the mRNA (initial transcriptionproduct) for the longest. Considering the ease of synthesis,antigenicity and other issues, for example, oligonucleotides consistingof about 15 nucleotides or more, preferably about 15 to about 30nucleotides, can be mentioned.

The target sequence for the antisense nucleic acid may be any sequencethat inhibits the translation of the NCS protein gene or a functionalfragment thereof by being hybridized to the antisense nucleic acid, andmay be the entire sequence or a partial sequence of mRNA, or the intronportion of the initial transcription product; when an oligonucleotide isused as the antisense nucleic acid, it is desirable that the targetsequence be located in a position from the 5′ terminus of the mRNA ofthe NCS protein gene to the C terminus of the coding region thereof.

Furthermore, the antisense nucleic acid may be not only capable ofhybridizing to a transcription product of the NCS protein gene toinhibit its translation, but also binding to the NCS protein gene in theform of double-stranded DNA to form a triple-strand (triplex) andinhibit the transcription to mRNA.

Another example of the substance that suppresses the expression of theNCS protein gene is a ribozyme capable of specifically cleaving atranscription product, specifically mRNA or initial transcriptionproduct, of the NCS protein gene in the coding region (including theintron portion in the case of initial transcription product). “Aribozyme” refers to an RNA possessing enzymatic activity to cleavenucleic acids. Because it has recently been shown that an oligo-DNAhaving the nucleotide sequence of the enzymatic activity site alsopossesses nucleic acid cleavage activity, this term is herein used tomean a concept including DNA, as long as it possesses sequence specificnucleic acid cleavage activity. The most versatile ribozyme isself-splicing RNA, found in infectious RNAs such as those of viroid andvirosoid; this self-splicing RNA is known as hammerhead type, hairpintype and the like. When ribozyme is used in the form of an expressionvector comprising a DNA that encodes the same, a hybrid ribozyme whereina sequence modified from tRNA is further linked to promote localizationto cytoplasm may be used [Nucleic Acids Res., 29(13): 2780-2788 (2001)].

A still another example of the substance that suppresses the expressionof the NCS protein gene is a decoy nucleic acid. A decoy nucleic acidrefers to a nucleic acid molecule that mimics a region to which atranscription regulatory factor binds; the decoy nucleic acid, which isthe substance that suppresses the expression of the NCS protein gene,can be a nucleic acid molecule that mimics a region to which atranscription activation factor for the NCS protein gene binds.

Examples of the decoy nucleic acid include oligonucleotides modified tomake them unlikely to undergo degradation in a body, such asoligonucleotides having a thiophosphoric diester bond wherein an oxygenatom in the phosphoric diester bond moiety is replaced with a sulfuratom (S-oligo), and oligonucleotides wherein the phosphoric diester bondis replaced with an uncharged methyl phosphate group, and the like.Although the decoy nucleic acid may completely match with the region towhich a transcription activation factor binds, the degree of matchingmay be such that the binding ability of the decoy nucleic acid to thetranscription activation factor for the NCS protein gene is retained.The length of the decoy nucleic acid is not subject to limitation, aslong as the transcription activation factor binds thereto. The decoynucleic acid may comprise a repeat of the same region.

Still another example of the substance that suppresses the expression ofthe NCS protein gene is a double-stranded oligo-RNA, i.e. siRNA, whichis complementary to a partial sequence (including the intron portion inthe case of an initial transcription product) in the coding region of atranscription product, specifically, the mRNA or initial transcriptionproduct, of the NCS protein gene. It has been known that so-called RNAinterference (RNAi), which is a phenomenon that if short double strandedRNA is introduced into cells, mRNA complementary to the RNA is degraded,occurs in nematodes, insects, plants and the like; recently, it has beenfound that this phenomenon also occurs in animal cells [Nature,411(6836): 494-498 (2001)], which is drawing attention as an alternativetechnique to ribozymes. The siRNA used may be internally synthesized asdescribed below, and a commercially available one may be used.

The antisense oligonucleotide and ribozyme can be prepared bydetermining the target sequence for a transcription product,specifically the mRNA or initial transcription product, of the NCSprotein gene on the basis of the cDNA sequence or genomic DNA sequenceof the NCS protein gene, and by synthesizing a sequence complementarythereto using a commercially available automated DNA/RNA synthesizer(Applied Biosystems Company, Beckman Instruments Company and the like).A decoy nucleic acid and siRNA can be prepared by synthesizing a sensestrand and an antisense strand in an automated DNA/RNA synthesizer,respectively, denaturing the strands in an appropriate annealing buffersolution at about 90 to about 95° C. for about 1 minute, and thenannealing the strands at about 30 to about 70° C. for about 1 to about 8hours. A longer double-stranded polynucleotide can be prepared bysynthesizing a complementary oligonucleotide chain in alternativeoverlaps, annealing them, and then ligating them with ligase.

Another example of the substance that suppresses the expression of theNCS protein gene is an antibody against the NCS protein. The antibodymay be a polyclonal antibody or a monoclonal antibody, and can beprepared by a well-known immunological technique. The antibody may alsobe a fragment of an antibody (e.g., Fab, F(ab′)₂), or a recombinantantibody (e.g., single-chain antibody). Furthermore, the nucleic acidthat encodes the antibody (one functionally linked to a nucleic acidhaving promoter activity) is also preferable as the substance thatsuppresses the expression of the NCS protein gene.

The polyclonal antibody can be acquired by, for example, subcutaneouslyor intraperitoneally administering the NCS protein or a fragment thereof(as required, may be prepared as a complex crosslinked to a carrierprotein such as bovine serum albumin or KLH (keyhole limpet hemocyanin))as the antigen, along with a commercially available adjuvant (e.g.,Freund's complete or incomplete adjuvant) to an animal about 2 to 4times at intervals of 2 to 3 weeks (the antibody titer of partiallydrawn serum has been determined by a known antigen-antibody reaction andits elevation has been confirmed in advance), collecting whole bloodabout 3 to about 10 days after final immunization, and purifying theantiserum. As the animal to receive the antigen, mammals such as rats,mice, rabbits, goat, guinea pigs, and hamsters can be mentioned.

The monoclonal antibody can be prepared by, for example, a cell fusionmethod (e.g., Takeshi Watanabe, Saibou Yugouhou No Genri To MonokuronaruKoutai No Sakusei, edited by Akira Taniuchi and Toshitada Takahashi,“Monokuronaru Koutai To Gan—Kiso To Rinsho—”, pages 2-14, Science ForumShuppan, 1985). For example, the factor is administered subcutaneouslyor intraperitoneally along with a commercially available adjuvant to amouse 2 to 4 times, and about 3 days after final administration, thespleen or lymph nodes are collected, and leukocytes are collected. Theseleukocytes and myeloma cells (e.g., NS-1, P3X63Ag8 and the like) arecell-fused to obtain a hybridoma that produces a monoclonal antibodyagainst the factor. This cell fusion may be performed by the PEG method[J. Immunol. Methods, 81(2): 223-228 (1985)], or by the voltage pulsemethod [Hybridoma, 7(6): 627-633 (1988)]. A hybridoma that produces thedesired monoclonal antibody can be selected by detecting an antibodythat binds specifically to the antigen from the culture supernatantusing a widely known EIA or RIA method and the like. Cultivation of thehybridoma that produces the monoclonal antibody can be performed invitro, or in vivo such as in mouse or rat ascitic fluid, preferably inmouse ascitic fluid, and the antibody can be acquired from the culturesupernatant of the hybridoma and the ascitic fluid of the animal,respectively.

However, in view of therapeutic efficacy and safety in humans, theantibody of the present invention may be a chimeric antibody or ahumanized or human type antibody. The chimeric antibody can be preparedwith reference to, for example, “Jikken Igaku (extra issue), Vol. 6, No.10, 1988”, Japanese Patent Kokoku Publication No. HEI-3-73280 and thelike. The humanized antibody can be prepared with reference to, forexample, Japanese Patent Kohyo Publication No. HEI-4-506458, JapanesePatent Kokai Publication No. SHO-62-296890 and the like. The humanantibody can be prepared with reference to, for example, “NatureGenetics, Vol. 15, p. 146-156, 1997”, “Nature Genetics, Vol. 7, p.13-21, 1994”, Japanese Patent Kohyo Publication No. HEI-4-504365,International Patent Application Publication No. WO94/25585, “NikkeiScience, June issue, pp. 40 to 50, 1995”, “Nature, Vol. 368, pp.856-859, 1994”, Japanese Patent Kohyo Publication No. HEI-6-500233 andthe like.

The substance that regulates the expression or function of the NCSprotein gene can also be a substance that suppresses a function of theNCS protein gene.

Although the substance that suppresses a function of the NCS proteingene is not subject to limitation, as long as it is capable ofinterfering with an action of the NCS protein gene, it is important thatthe substance be capable of specifically acting on the target moleculeto minimize the adverse effect on other genes and proteins. Examples ofthe substance that specifically suppresses a function of the NCS proteingene include a dominant negative mutant of the NCS protein and a nucleicacid that encodes the mutant (one functionally linked to a nucleic acidhaving promoter activity).

A dominant negative mutant of the NCS protein refers to a mutant havingthe activity thereof reduced as a result of mutagenesis to the NCSprotein. The dominant negative mutant can indirectly inhibit theactivity of natural NCS protein by competing with the protein. Thedominant negative mutant can be prepared by introducing a mutation to anucleic acid that encodes the NCS protein gene. Examples of, themutation include amino acid mutations (e.g., deletion, substitution, andaddition of one or more amino acids) in a site of EF hand motif,myristoylation site and sites other than these sites that result in adecrease in the function responsible for the sites. The mutation can beintroduced by a method known per se using PCR or a commonly known kit.

The substance that regulates the function of NCS protein also includesthe compounds described above and salts thereof.

Provided that the substance that suppresses the expression of the NCSprotein gene is a nucleic acid molecule, the regulator of the presentinvention can have an expression vector that encodes the nucleic acidmolecule as the active ingredient thereof. The expression vector is anoligonucleotide or polynucleotide that encodes the above-describednucleic acid molecule, and must be functionally linked to a promotercapable of exhibiting promoter activity in the cells of the recipientmammal. Any promoter capable of functioning in the recipient mammal canbe used; examples include viral promoters such as the SV40-derived earlypromoter, cytomegalovirus LTR, Rous sarcoma virus LTR, MoMuLV-derivedLTR, and adenovirus-derived early promoter, and mammalian structuralprotein gene promoters such as the β-actin gene promoter, PGK genepromoter, and transferrin gene promoter, and the like.

The expression vector preferably comprises a transcription terminationsignal, that is, a terminator region, downstream of the oligo(poly)nucleotide that encodes the nucleic acid molecule. The expressionvector may further comprise a selection marker gene for selectingtransformant cells (genes that confer resistance to drugs such astetracycline, ampicillin, kanamycin, hygromycin, and phosphinothricin,gene that compensate for auxotrophic mutation, and the like).

Although the basic backbone vector used as the expression vector is notsubject to limitation, vectors suitable for administration to mammalssuch as humans include viral vectors such as retrovirus, adenovirus,adeno-associated virus, herpesvirus, vaccinia virus, poxvirus,poliovirus, Sindbis virus, and Sendai virus. Adenovirus has advantageousfeatures, including the very high efficiency of gene introduction andpossibility of introduction to non-dividing cells. Because incorporationof the introduced gene to host chromosome is very rare, gene expressionis transient, usually lasting for about 4 weeks. In view of thesustainability of therapeutic effect, it is also preferable to useadeno-associated virus, which offers relatively high gene transductionefficiency, which can be introduced to non-dividing cells, and which canbe incorporated in chromosomes via a inverted terminal repeat sequence(ITR).

The substance that regulates the expression or function of the NCSprotein can also be the NCS protein-targeting drug described above, andcan be, for example, atorvastatin, pimozide, bifonazole, flunarizine,fendiline, chloperastine, bepridil, raloxifene hydrochloride,benzbromarone, prazepam, clotiazepam, suloctidil, benzethonium,bicaltamide, benzthiazide, minaprine, trifluoperazine, chlorprothixene,pimethixene, flupentixol, clofazimine, loxapine, rescinnamine,syrosingopine, dihydroergocornine mesylate, dihydro-α-ergocryptinemesylate, dihydro-β-ergocryptine mesylate, dihydroergocristine mesylateor stanozolol, or a derivative thereof capable of binding to the NCSprotein (described later), or a salt thereof.

Regulator I can comprise any carrier, for example, a pharmaceuticallyacceptable carrier, in addition to a substance that regulates theexpression or function of the NCS protein gene.

Examples of the pharmaceutically acceptable carrier include, but are notlimited to, excipients such as sucrose, starch, mannitol, sorbitol,lactose, glucose, cellulose, talc, calcium phosphate, and calciumcarbonate; binders such as cellulose, methylcellulose,hydroxypropylcellulose, polypropylpyrrolidone, gelatin, gum arabic,polyethylene glycol, sucrose, and starch; disintegrants such as starch,carboxymethylcellulose, hydroxypropylstarch, sodium-glycol-starch,sodium hydrogen carbonate, calcium phosphate, and calcium citrate;lubricants such as magnesium stearate, Aerosil, talc, and sodium laurylsulfate; flavoring agents such as citric acid, menthol, glycyrrhizinammonium salt, glycine, and orange powder; preservatives such as sodiumbenzoate, sodium hydrogen sulfite, methyl paraben, and propyl paraben;stabilizers such as citric acid, sodium citrate, and acetic acid;suspending agents such as methylcellulose, polyvinylpyrrolidone, andaluminum stearate; dispersing agents such as surfactants; diluents suchas water, physiological saline, and orange juice; base waxes such ascacao fat, polyethylene glycol, and kerosene, and the like.

Preparations suitable for oral administration include liquids comprisingan effective amount of substance dissolved in a diluent such as water,physiological saline, or orange juice, capsules, sachets or tabletscomprising an effective amount of substance in the form of solid orgranules, suspensions comprising an effective amount of substancesuspended in an appropriate dispersant, emulsions comprising a solutionof an effective amount of substance dispersed in an appropriatedispersant and the like.

Preparations suitable for parenteral administration (e.g., subcutaneousinjection, intramuscular injection, topical injection, intraperitonealinjection, and the like) include aqueous and non-aqueous isotonicsterile injection liquids, which may comprise an antioxidant, a buffersolution, a bacteriostatic agent, an isotonizing agent and the like.Other examples are aqueous and non-aqueous sterile suspensions, whichmay comprise a suspending agent, a solubilizer, a thickening agent, astabilizer, an antiseptic and the like. The preparation can be includedin a container in a unit dose or multiple doses like an ampoule or vial.It is also possible to lyophilize the active ingredient and apharmaceutically acceptable carrier and preserve them in a state thatonly requires dissolving or suspending in a suitable sterile vehicleimmediately before use.

The dose of regulator I varies depending on the activity and kind of theactive ingredient, severity of the disease, the animal species to be theadministration subject, drug acceptability, body weight and age of theadministration subject, and the like, it is generally about 0.001 toabout 500 mg/kg a day for an adult based on the amount of the activeingredient.

Regulator I enables the regulation, for example, suppression orpromotion, of an action associated with NCS protein-targeting drug.Hence, regulator I is useful for the prophylaxis and treatment of adisease associated with NCS protein-targeting drug, and as aninvestigational reagent for the disease, and the like.

3.2. Regulator of a Function Associated with the NCS Protein Gene(Regulator II)

The present invention provides a regulator of a function associated withthe NCS protein gene, which comprises NCS protein-targeting drug.

This regulator is optionally abbreviated as “regulator II.”

The NCS protein-targeting drug can be as described above, and can be,for example, atorvastatin, pimozide, bifonazole, flunarizine, fendiline,chloperastine, bepridil, raloxifene hydrochloride, benzbromarone,prazepam, clotiazepam, suloctidil, benzethonium, bicaltamide,benzthiazide, minaprine, trifluoperazine, chlorprothixene, pimethixene,flupentixol, clofazimine, loxapine, rescinnamine, syrosingopine,dihydroergocornine mesylate, dihydro-α-ergocryptine mesylate,dihydro-β-ergocryptine mesylate, dihydroergocristine mesylate orstanozolol, or a derivative thereof capable of binding to the NCSprotein (described later), or a salt thereof.

Regulator II can comprise any carrier, for example, a pharmaceuticallyacceptable carrier, in addition to NCS protein-targeting drug. The doseof regulator II is the same as that of regulator I.

Regulator II enables the regulation, for example, suppression orpromotion, of a function associated with the NCS protein gene. Hence,regulator II is useful for the prophylaxis and treatment of a diseaseassociated with the NCS protein gene, and as an investigational reagentfor the disease, and the like.

4. Derivative Production Method and Product Obtained by the Method

The present invention provides methods of producing a drug derivative,comprising derivatizing a drug so as to be able to regulate theexpression or function of the NCS protein gene, and the productsobtained thereby. The production methods of the present invention can beroughly divided into a method of producing a drug derivative capable ofregulating an action associated with NCS protein-targeting drug (e.g.,central nervous action), and a method of producing a drug derivativecapable of regulating a function associated with the NCS protein gene,from the viewpoint of the kinds of action and function of the derivativeobtained. The individual production methods are hereinafter described indetail.

4.1. Production Method of a Drug Derivative Capable of Regulating anAction Associated with NCS Protein-Targeting Drug (Production Method I)

The present invention provides a method of producing a drug derivativecapable of regulating an action associated with NCS protein-targetingdrug, comprising derivatizing a drug so as to be able to regulate theexpression or function of the NCS protein gene.

This production method is optionally abbreviated as “production methodI.”

Derivatization means that a compound obtained by replacing a particularatom or group in a lead compound with another atom or group, or acompound obtained by subjecting a lead compound to an addition reaction,is virtually or actually synthesized. For example, the lead compound canbe an NCS protein-targeting drug. The NCS protein-targeting drug is notparticularly limited, and can be, for example, statin drugs havinganti-central nervous action.

The derivatization of NCS protein-targeting drug can be performed sothat the regulatory capability for the expression or function of the NCSprotein gene is retained, and as required, in view of other propertiesof the derivative obtained, such as hydrophilicity/liphophilicity,stability, dynamics, bioavailability, toxicity and the like. Thederivatization of NCS protein-targeting drug can be performed so that,for example, the regulatory capability for the expression or function ofthe NCS protein gene can be increased. The derivatization of NCSprotein-targeting drug can also be performed so that a functionassociated with the NCS protein gene can be regulated.

The derivatization of NCS protein-targeting drug such that theregulatory capability for the expression or function of the NCS proteingene is retained can be performed on the basis of, for example, SBDD(structure-based drug design: SBDD) and CADD (computer-aided drugdesign). Examples of the design include virtual screening, de novodesign, pharmacophore analysis, QSAR (quantitative structure activityrelationship) and the like. If information on the steric structure ofthe protein itself or the target site of the protein is required duringsuch designing, information on the steric structure is used providedthat the steric structure is known by a structural analytical techniquesuch as NMR, X-ray crystallographic analysis, or synchrotron radiationanalysis. If the steric structure is unknown, information obtained by astructural predictive method such as the homology method or thethreading method is used. In virtual screening, a program known per seis used; examples of the program include DOCK (Kuntz, I. D. et al.,Science, 1992, 257, 1078), Gold (Jones, G. et al., J. Mol. Biol., 1995,245, 43), FlexX (Rarey, M. et al., J. Mol. Biol., 1996, 261, 470),AtutoDock (Morris, G. M. et al., J. Comput. Chem., 1998, 19, 1639), ICM(Abagyan, R. A. et al., J. Comput. Chem., 1994, 15, 488) and the like.

The derivatization of NCS protein-targeting drug such that theregulatory capacity for the expression or function of the NCS proteingene is retained can also be performed on the basis of, for example,biological verification. In this case, for example, the above-describedmethodologies I to IV can be used. Furthermore, the above-describedmethod such as SBDD or CADD, and biological verification may be used incombination.

A particular atom in the NCS protein-targeting drug, which issubstituted for the production of derivative, is not limited as long asit is present in a lead compound and, for example, a hydrogen atom, ahalogen atom (e.g., a fluorine atom, a chlorine atom, a bromine atom, aniodine atom), an oxygen atom, a sulfur atom, a nitrogen atom, a carbonatom and the like can be mentioned.

The particular group in the NCS protein-targeting drug, which issubstituted for the production of derivative, is not limited as long asit is present in the NCS protein-targeting drug and may be, for example,a group having a molecular weight of 1 to 500, preferably 1 to 300, morepreferably 1 to 200, most preferably 1 to 100. As the particular group,for example, an optionally substituted C₁-C₈ hydrocarbon group, anoptionally substituted C₁-C₈ acyl group, an optionally substitutedaromatic or non-aromatic C₃-C₁₄ hydrocarbon ring group, an optionallysubstituted aromatic or non-aromatic C₃-C₁₄ heterocyclic group, an aminogroup, an amino group mono- or di-substituted by an alkyl group having 1to 4 carbon atoms or an acyl group having 2 to 8 carbon atoms, anamidino group, a carbamoyl group, a carbamoyl group mono- ordi-substituted by an alkyl group having 1 to 4 carbon atoms, a sulfamoylgroup, a sulfamoyl group mono- or di-substituted by an alkyl grouphaving 1 to 4 carbon atoms, a carboxyl group, an alkoxycarbonyl grouphaving 2 to 8 carbon atoms, a hydroxy group, an alkoxyl group having 1to 6 carbon atoms and optionally substituted by 1 to 3 halogen atoms, analkenyloxy group having 2 to 5 carbon atoms and optionally substitutedby 1 to 3 halogen atoms, a cycloalkyloxy group having 3 to 7 carbonatoms, an aralkyloxy group having 7 to 9 carbon atoms, an aryloxy grouphaving 6 to 14 carbon atoms, a thiol group, an alkylthio group having 1to 6 carbon atoms and optionally substituted by 1 to 3 halogen atoms, anaralkylthio group having 7 to 9 carbon atoms, an arylthio group having 6to 14 carbon atoms, a sulfo group, a cyano group, an azido group, anitro group, a nitroso group and the like can be mentioned.

The optionally substituted C₁-C₈ hydrocarbon group may be, for example,an optionally substituted C₁-C₈ alkyl group, an optionally substitutedC₂-C₈ alkenyl group or an optionally substituted C₂-C₈ alkynyl group.

The C₁-C₈ alkyl group of the optionally substituted C₁-C₈ alkyl groupmay be straight chain or branched chain, and preferably has 1 to 6carbon atoms. For example, methyl, ethyl, propyl, isopropyl, butyl,isobutyl, sec-butyl, tert-butyl and the like can be mentioned.

The C₂-C₈ alkenyl group of the optionally substituted C₂-C₈ alkenylgroup may be straight chain or branched chain, and preferably has 2 to 6carbon atoms. For example, ethenyl, 1-propenyl, 2-propenyl,2-methyl-1-propenyl, 1-butenyl, 2-butenyl, 3-butenyl and the like can bementioned.

The C₂-C₈ alkynyl group of the optionally substituted C₂-C₈ alkynylgroup may be straight chain or branched chain, and preferably has 2 to 6carbon atoms. For example, ethynyl, 1-propynyl, 2-propynyl, 1-butynyl,2-butynyl, 3-butynyl and the like can be mentioned.

The C₁-C₈ acyl group of the optionally substituted C₁-C₈ acyl group maybe straight chain or branched chain, and preferably has 2 to 6 carbonatoms. For example, formyl, acetyl, propanoyl, butanoyl,2-methylpropanoyl and the like can be mentioned.

The aromatic C₃-C₁₄ hydrocarbon ring group of the optionally substitutedaromatic C₃-C₁₄ hydrocarbon ring group may be monocyclic, bicyclic ortricyclic, and preferably has 3 to 12 carbon atoms. For example, phenyland naphthyl can be mentioned.

The non-aromatic C₃-C₁₄ hydrocarbon ring group of the optionallysubstituted non-aromatic C₃-C₁₄ hydrocarbon ring group may be saturatedor unsaturated, monocyclic, bicyclic or tricyclic, and preferably has 3to 12 carbon atoms. For example, a cycloalkyl group (e.g., cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl), acycloalkenyl group (e.g., 2-cyclopenten-1-yl, 3-cyclopenten-1-yl,2-cyclohexen-1-yl, 3-cyclohexen-1-yl), a cycloalkadienyl group (e.g.,2,4-cyclopentadien-1-yl, 2,4-cyclohexadien-1-yl, 2,5-cyclohexadien-1-yl)and the like can be mentioned.

The aromatic C₃-C₁₄ heterocyclic group of the optionally substitutedaromatic C₃-C₁₄ heterocyclic group is a monocyclic, bicyclic ortricyclic aromatic heterocyclic group containing, as a ring-constitutingatom besides carbon atoms, 1 to 5 heteroatoms selected from an oxygenatom, a sulfur atom and a nitrogen atom, and preferably has 3 to 12carbon atoms. As examples of monocyclic aromatic C₃-C₁₄ heterocyclicgroup, furyl, thienyl, pyrrolyl, oxazolyl, isoxazolyl, thiazolyl,isothiazolyl, imidazolyl, pyrazolyl, oxadiazolyl, furazanyl,thiadiazolyl, triazolyl, tetrazolyl, pyridyl, pyrimidinyl, pyridazinyl,pyrazinyl, triazinyl and the like can be mentioned. As examples ofbicyclic or tricyclic aromatic heterocyclic group, benzo furanyl,isobenzofuranyl, benzo[b]thienyl, indolyl, isoindolyl, 1H-indazolyl,benzimidazolyl, benzoxazolyl, benzothiazolyl, 1H-benzotriazolyl,quinolyl, isoquinolyl, cinnolyl, quinazolyl, quinoxalinyl, phthalazinyl,naphthyridinyl, purinyl, pteridinyl, carbazolyl, α-carbolinyl,β-carbolinyl, γ-carbolinyl, acrydinyl, phenoxazinyl, phenothiazinyl,phenazinyl, phenoxathiinyl, thianthrenyl, indolizinyl,pyrrolo[1,2-b]pyridazinyl, pyrazolo[1,5-a]pyridyl,imidazo[1,2-a]pyridyl, imidazo[1,5-a]pyridyl, imidazo[1,2-b]pyridazinyl,imidazo[1,2-a]pyrimidinyl, 1,2,4-triazolo[4,3-a]pyridyl,1,2,4-triazolo[4,3-b]pyridazinyl and the like can be mentioned.

The non-aromatic C₃-C₁₄ heterocyclic group of the optionally substitutednon-aromatic C₃-C₁₄ heterocyclic group is a monocyclic, bicyclic ortricyclic, saturated or unsaturated heterocyclic group containing, as aring-constituting atom besides carbon atoms, 1 to 5 heteroatoms selectedfrom an oxygen atom, a sulfur atom and a nitrogen atom, and preferablyhas 3 to 12 carbon atoms. For example, oxylanyl, azetidinyl, oxetanyl,thietanyl, pyrrolidinyl, tetrahydrofuryl, tetrahydropyranyl,morpholinyl, thiomorpholinyl, piperazinyl, pyrrolidinyl, piperidino,morpholino, thiomorpholino and the like can be mentioned.

The kind of the substituent in any group optionally substituted can bethe same as the particular group in NCS protein-targeting drug(described above), which is substituted for producing the derivative.

The number of particular atoms or groups in NCS protein-targeting drug,which is substituted for producing the derivative is any number, as longas the derivative produced is capable of regulating the expression orfunction of the NCS protein gene, for example, as long as it is capableof binding to the NCS protein, and can be, for example, 1 to 10,preferably 1 to 5, more preferably 1 to 3, further more preferably 1 to2, most preferably 1.

The kind of a particular atom or group used for substitution (i.e., anatom or group introduced to the substitution site) can be the same asthe particular atom or group in NCS protein-targeting drug, which issubstituted for producing the derivative.

The atom or group added to NCS protein-targeting drug for producing thederivative (i.e., an atom or group used in the addition reaction) is anatom permitting an addition reaction, for example, an atom such as thehydrogen atom or the halogen atom, or a group capable of acting as anucleophile or electrophile, out of the particular atoms or groups inNCS protein-targeting drug (described above), which is substituted forproducing the derivative.

The number of atoms or groups added to NCS protein-targeting drug forproducing the derivative is any number, as long as the derivativeproduced is capable of regulating the expression or function of the NCSprotein gene, for example, as long as it is capable of binding to theNCS protein, and can be, for example, less than 6, preferably less than4, more preferably less than 2.

The production method I is useful for, for example, the development ofprophylactic or therapeutic agents for diseases associated with NCSprotein-targeting drug (for example, central nervous diseases),investigational reagents for the diseases, and the like.

4.2. Production Method of a Drug Derivative Capable of Regulating aFunction Associated with the NCS Protein Gene (Production Method II)

The present invention provides a method of producing a drug derivativecapable of regulating a function associated with the NCS protein gene,comprising derivatizing a drug so as to be able to regulate the abilityof the drug to bind to NCS protein or a mutant protein thereof.

This production method is optionally abbreviated as “production methodII.”

The derivatization of NCS protein-targeting drug can be performed sothat the binding capability for the NCS protein or a mutant proteinthereof is retained, and as required, in view of other properties of thederivative obtained, such as hydrophilicity/liphophilicity, stability,dynamics, bioavailability, toxicity and the like. The derivatization ofthe drug can be performed so that, for example, the binding capabilitycan be increased.

The derivatization of a drug such that the binding capability isretained can be performed on the basis of, for example, SBDD and CADD.

The derivatization of a drug such that the binding capability isretained can also be performed on the basis of, for example, biologicalverification. In this case, the derivatization can be performed as issimilar to, for example, the above-described methodologies IV.Furthermore, the above-described method such as SBDD or CADD, andbiological verification may be used in combination.

The particular atom or group in lead compound, which is substituted forproducing the derivative, and the number of the atoms or groups can bethe same as those described above. The particular atom or group used forsubstitution (i.e., the atom and group introduced to the substitutionsite), the atom or group added for producing the derivative (i.e., theatom or group used for additive reaction), and the number of the atomsor groups are the same as those described above.

The production method II is useful for the development of prophylacticor therapeutic agents for diseases associated with NCS protein gene,investigational reagents for the diseases, and the like.

4.3. Product Obtained by the Derivative Production Method

The present invention provides a product obtained by the above-describedmethods I and II.

The product provided by the above-described production method can be aderivative of an NCS protein-targeting drug, which is obtained by theproduction method of the present invention, and a pharmacological actionregulator comprising the derivative (described above).

A product provided by the above-described production method is usefulfor, for example, the prophylaxis or treatment of diseases associatedwith NCS protein-targeting drug, or diseases associated with the NCSprotein gene, or as investigational reagents for the disease, and thelike.

5. Complex and Production Method Thereof

The present invention provides a complex comprising a drug and NCSprotein or a mutant protein thereof.

The drug can be the above-described NCS protein-targeting drug, andincludes, for example, atorvastatin, pimozide, bifonazole, flunarizine,fendiline, chloperastine, bepridil, raloxifene hydrochloride,benzbromarone, prazepam, clotiazepam, suloctidil, benzethonium,bicaltamide, benzthiazide, minaprine, trifluoperazine, chlorprothixene,pimethixene, flupentixol, clofazimine, loxapine, rescinnamine,syrosingopine, dihydroergocornine mesylate, dihydro-α-ergocryptinemesylate, dihydro-β-ergocryptine mesylate, dihydroergocristine mesylateand stanozolol, and a derivative thereof capable of binding to the NCSprotein, and the like.

The present invention also provides a method of producing a complexcomprising a drug and NCS protein or a mutant thereof, which comprisesbringing the drug and the NCS protein or a mutant thereof into contactwith each other. This contact can be performed by, for example, mixingthe drug and the protein in solution.

The complex of the present invention and the production method of thecomplex can be useful in, for example, performing the screening methodsof the present invention or the production method of the derivative ofthe present invention, or in cases where the complex is structurallyanalyzed to extensively investigate the mode of interaction between adrug and a protein, and the like.

6. Kit

The present invention provides a kit comprising a drug or a saltthereof.

In one embodiment, the kit of the present invention comprises thefollowing (i) and (ii):

-   (i) a drug or a salt thereof;-   (ii) NCS protein or a mutant thereof, a nucleic acid that encodes    the protein, an expression vector comprising the nucleic acid, cells    enabling a measurement of the expression of the NCS protein gene, or    an expression vector comprising the transcription regulatory region    of the NCS protein gene and a reporter gene functionally linked to    the region.

Provided that the kit of the present invention comprises the protein,the protein is not in the form of a complex with the drug.

The expression vector, the cells enabling a measurement of theexpression of the NCS protein gene, the transcription regulatory regionof the NCS protein gene, and the reporter gene functionally linked tothe region, are the same as those described above (see, e.g., “2.Screening method, and product obtained by the method”).

The above-described kit of the present invention can be useful in, forexample, performing the screening methods of the present invention, theproduction method of the derivative of the present invention, and theproduction method of the complex of the present invention, and the like.

7. Determination Method and Determination Kit for the Onset or Risk ofOnset of Disease

The present invention provides determination methods and determinationkits for the onset or risk of onset of a specified disease. Thedetermination methods and determination kits of the present inventioncan be roughly divided into a determination method and determination kitbased on measurement of the expression level, and a determination methodand determination kit based on identification of the polymorphism, fromthe viewpoint of the subject to be measured. Furthermore, they can beclassified into a determination method and determination kit for theonset or risk of onset of a disease associated with NCSprotein-targeting drug (for example, central nervous disease), and adetermination method and determination kit for the onset or risk ofonset of a disease associated with the NCS protein gene, from theviewpoint of the disease for which a determination of the onset or riskof onset is desired. The individual determination methods anddetermination kits are hereinafter described in detail.

7.1. Determination Method and Determination Kit for the Onset or Risk ofOnset of Disease on the Basis of Measurement of the Expression Level ofthe NCS Protein Gene

7.1.1. Determination Method for the Onset or Risk of Onset of DiseaseAssociated with NCS Protein-Targeting Drug on the Basis of Measurementof the Expression Level of the NCS Protein Gene (Determination Method I)

The present invention provides a determination method for the onset orrisk of onset of a disease associated with NCS protein-targeting drug,which comprises measuring the expression level of the NCS protein gene.

This determination method is optionally abbreviated as “determinationmethod I.”

In one embodiment, determination method I comprises the following steps(a) and (b):

-   (a) a step for measuring the expression level of the NCS protein    gene in a biological sample collected from an animal;-   (b) a step for evaluating the onset or likelihood of onset of a    disease associated with NCS protein-targeting drug on the basis of    the expression level of the NCS protein gene.

The methodology comprising the above-described steps (a) to (b) isoptionally abbreviated as “methodology V.”

In step (a) of methodology V, the expression level of the NCS proteingene in a biological sample collected from an animal is measured.Although the animal is not particularly limited, mammal such aslaboratory animals such as mice, rats, hamsters, guinea pigs, andrabbits, domestic animals such as swine, bovine, goat, horses, andsheep, companion animals such as dogs and cats, and primates such asmonkeys, orangutans, chimpanzees, and humans can be mentioned.

The biological sample may be any sample containing a tissue expressingthe NCS protein gene. The tissue expressing the NCS protein gene is asdescribed above. The expression level of the NCS protein gene can bemeasured by a method known per se with a product, for example, atranscription product or translation product, of the NCS protein gene,as the subject. In step (b) of methodology V, a determination is madewhether or not the animal is suffering from a disease associated withNCS protein-targeting drug on the basis of the expression level of theNCS protein gene. Specifically, first, the measured expression level ofthe NCS protein gene is compared with the expression level of the NCSprotein gene in an animal that has not contracted the disease associatedwith NCS protein-targeting drug (e.g., a normal animal). This comparisonof expression level is preferably performed on the basis of the presenceor absence of a significant difference. The expression level of the NCSprotein gene in an animal that has not contracted the disease associatedwith NCS protein-targeting drug can be determined by a method known perse.

Next, on the basis of the result of the comparison of the expressionlevel of the NCS protein gene, a judgement is made whether or not theanimal is possibly suffering from a disease associated with NCSprotein-targeting drug, or is likely or unlikely to suffer from thedisease in the future. It is known that in animals that have contracteda particular disease, a change in the expression of the gene associatedwith the disease is often observed. It is also known that prior to theonset of a particular disease, a change in the expression of theparticular gene is often observed. Hence, by analyzing the expressionlevel of the NCS protein gene, it is possible to determine the onset orlikelihood of onset of the disease associated with NCS protein-targetingdrug.

Determination method I enables a determination of the presence orabsence of a disease associated with NCS protein-targeting drug, or thelikelihood of contracting the disease. Hence, determination method I isuseful for, for example, the easy and early detection of the disease,and the like.

7.1.2. Determination Kit for the Onset or Risk of Onset of DiseaseAssociated with NCS Protein-Targeting Drug on the Basis of Measurementof Expression Level of the NCS Protein Gene (Determination Kit I)

The present invention provides a determination kit that enables the easyconduct of determination method I.

This determination kit is optionally abbreviated as “determination kitI.”

In one embodiment, determination kit I comprises the following (i) and(ii):

-   (i) a means capable of measuring the expression level of the NCS    protein gene;-   (ii) a medium recording the relationship between a disease    associated with NCS protein-targeting drug and the expression level    of the NCS protein gene.

The means capable of measuring the expression level of the NCS proteingene is not subject to limitation, as long as it allows a quantitationof the expression level of the NCS protein gene; for example, such meansare roughly divided into means capable of quantifying NCS protein (forexample, antibody, or NCS protein-targeting drug), and means capable ofquantifying a transcription product of the NCS protein gene (forexample, or nucleic acid probe, a pair of primers). The means may belabeled with a labeling substance. Provided that the means is notlabeled with a labeling substance, the determination kit of the presentinvention may further comprise the labeling substance. The labelingsubstance is the same as described above.

Determination kit I enables a determination of the presence or absenceof a disease associated with NCS protein-targeting drug, or thelikelihood of contracting the disease. Hence, determination kit I isuseful for, for example, the easy and early detection of the disease,and the like.

7.2. Determination Method and Determination Kit for the Risk of Onset ofDisease on the Basis of Identification of Polymorphism of the NCSProtein Gene

7.2.1. Determination Method for the Risk of Onset of Disease Associatedwith NCS Protein-Targeting Drug on the Basis of Identification ofPolymorphism of the NCS Protein Gene (Determination Method II)

The present invention provides a determination method for the risk ofonset of a disease associated with NCS protein-targeting drug; whichcomprises identifying the polymorphism of the NCS protein gene.

This determination method is optionally abbreviated as “determinationmethod II.”

In one embodiment, determination method II comprises the following steps(a) and (b):

-   (a) a step for identifying the polymorphism of the NCS protein gene    in a biological sample collected from an animal;-   (b) a step for evaluating the likelihood of the onset of a disease    associated with NCS protein-targeting drug on the basis of the type    of polymorphism.

The methodology comprising the above-described steps (a) to (b) isoptionally abbreviated as “methodology VI.”

In step (a) of methodology VI, the type of polymorphism of the NCSprotein gene is identified in a biological sample collected from ananimal. The animal is the same as described above.

Although the biological sample used may be one described with respect tomethodology V above, this methodology VI enables the use of any tissuecontaining genomic DNA, such as hair, nails, skin or mucosa, as thebiological sample. In view of the ease of procurement, burden on thehuman body and the like, the biological sample is preferably a sample ofhair, nails, skin, mucosa, blood, plasma, serum, saliva and the like.

A polymorphism of the NCS protein gene means a mutation found at afrequency in the nucleotide sequence of the genomic DNA comprising theNCS protein gene in a certain population, and can be one or more DNAsubstitutions, deletions, or additions (e.g., SNP, haplotype) in thegenomic DNA comprising the NCS protein gene, and a repeat, inversion,translocation and the like of the genomic DNA. Various types ofpolymorphism of the NCS protein gene are registered with knowndatabases, for example, H-Inv DB and the like. The type of polymorphismof the NCS protein gene used in this determination method is a mutationin a nucleotide sequence whose frequency differs between animalssuffering from a disease associated with NCS protein-targeting drug andnon-suffering animals, out of all types of polymorphism in the NCSprotein gene, and can be, for example, one that alters the expression ofthe NCS protein gene or alters a function associated with the NCSprotein gene (e.g., the ability of NCS protein to bind with NCSprotein-targeting drug). Such types of polymorphism can be determined bya method known per se such as linkage analysis.

The identification of the type of polymorphism can be performed by amethod known per se. For example, the RFLP (restriction fragment lengthpolymorphism) method, the PCR-SSCP (single-stranded DNA conformationpolymorphism) analysis method, the ASO (allele specific oligonucleotide)hybridization method, the TaqMan PCR method, the invader method and thelike can be used.

In step (b) of methodology VI, a determination of the likelihood ofcontracting a disease associated with NCS protein-targeting drug in ananimal is made on the basis of the type of polymorphism. It is knownthat animals susceptible to a particular disease often have a particulartype of polymorphism in the gene associated with the disease. Hence, itis possible to determine the likelihood of the onset of a diseaseassociated with NCS protein-targeting drug by polymorphism analysis.

Determination method II enables a determination of the likelihood ofcontracting a disease associated with NCS protein-targeting drug (e.g.,a central neurological disease). Hence, determination method II isuseful for the provision of an incentive for improving one's lifestylefor the purpose of preventing the disease, and the like.

7.2.2. Determination Kit for the Risk of Onset of Disease Associatedwith NCS Protein-Targeting Drug on the Basis of Identification ofPolymorphism of the NCS Protein Gene (Determination Kit II)

The present invention also provides a determination kit that enables theeasy conduct of determination method II.

This determination kit is optionally abbreviated as “determination kitII.”

In one embodiment, determination kit II comprises the following (i) and(ii):

-   (i) a means capable of identifying the polymorphism of the NCS    protein gene (for example, nucleic acid probe, or a pair of    primers);-   (ii) a medium recording the relationship between a disease    associated with an NCS protein gene and the polymorphism of the NCS    protein gene.

Determination kit II enables a determination of the likelihood ofcontracting a disease associated with NCS protein-targeting drug (e.g.,a central neurological disease). Hence, determination kit II is usefulfor the provision of an incentive for improving one's lifestyle for thepurpose of preventing the disease and the like.

7.2.3. Method of Determining the Risk of Onset of Disease Associatedwith the NCS Protein Gene on the Basis of Identification of Polymorphismof the NCS Protein Gene (Determination Method III)

The present invention provides a determination method for the risk ofonset of a disease associated with the NCS protein gene, which comprisesidentifying the polymorphism of the NCS protein gene.

This determination method is optionally abbreviated as “determinationmethod III.”

In one embodiment, determination method III comprises the followingsteps (a) and (b):

-   (a) a step for determining the type of the polymorphism of the NCS    protein gene in a biological sample collected from an animal;-   (b) a step for evaluating the likelihood of the onset of a disease    associated with the NCS protein gene on the basis of the type of    polymorphism.

In determination method III, the type of polymorphism used to determinethe risk of onset alters the ability of the NCS protein gene to bind toan NCS protein-targeting drug. Such a type of polymorphism can bedetermined by a method known per se such as binding assay.

The methodology comprising steps (a) and (b) above in determinationmethod III is the same as methodology VI except for the type ofpolymorphism of the NCS protein gene to be identified.

Determination method III enables a determination of the likelihood ofcontracting a disease associated with the NCS protein gene. Hence,determination method III is useful for the provision of an incentive forimproving one's lifestyle for the purpose of preventing the disease, andthe like.

7.2.4. Determination Kit for the Risk of Onset of Disease Associatedwith the NCS Protein Gene on the Basis of Identification of Polymorphismof the NCS Protein Gene (Determination Kit III)

The present invention also provides a determination kit that enables theeasy conduct of determination method III.

This determination kit is optionally abbreviated as “determination kitIII.”

In one embodiment, determination kit III comprises the following (i) and(ii):

-   (i) a means capable of identifying the polymorphism of the NCS    protein gene;-   (ii) a medium recording the relationship between a disease    associated with the NCS protein gene and the polymorphism of the NCS    protein gene.

In determination kit III, the type of polymorphism used to determine therisk of onset is one that alters the ability of NCS protein to bind withNCS protein-targeting drug. Such a type of polymorphism can bedetermined by a method known per se such as binding assay.

The constituents of determination kit III are the same as those ofdetermination kit II except for the type of polymorphism of the NCSprotein gene to be identified.

Determination kit III enables a determination of the likelihood ofcontracting a disease associated with the NCS protein gene. Hence,determination kit III is useful for the provision of an incentive forimproving one's lifestyle for the purpose of preventing the disease, andthe like.

8. Determination Method and Determination Kit for Susceptibility toDrugs

The present invention provides determination methods and determinationkits for susceptibility to a drug. The determination methods anddetermination kits of the present invention can be roughly divided intodetermination methods and determination kits based on measurement ofexpression level, and determination methods and determination kits basedon identification of polymorphism. Furthermore, they are classified intodetermination methods and determination kits for a disease associatedwith NCS protein-targeting drug, and determination methods anddetermination kits for a disease associated with the NCS protein gene,from the viewpoint of a disease for which a determination ofsusceptibility is desired. The individual determination methods anddetermination kits are hereinafter described in detail.

8.1. Determination Method and Determination Kit for Susceptibility toDrugs on the Basis of Measurement of the Expression Level of the NCSProtein Gene

8.1.1. Determination Method for Susceptibility to NCS Protein-TargetingDrug in Disease Associated with NCS Protein-Targeting Drug on the Basisof Measurement of the Expression Level of the NCS Protein Gene(Determination Method IV)

The present invention provides a determination method for susceptibilityto NCS protein-targeting drug in a disease associated with NCSprotein-targeting drug, which comprises measuring the expression levelof the NCS protein gene.

This determination method is optionally abbreviated as “determinationmethod IV.”

In one embodiment, determination method IV comprises the following steps(a) and (b):

-   (a) a step for measuring the expression level of the NCS protein    gene in a biological sample collected from an animal;-   (b) a step for predicting the effect of an NCS protein-targeting    drug on the basis of the expression level of the NCS protein gene.

The methodology comprising the above-described steps (a) to (b) isoptionally abbreviated as “methodology VII.”

Step (a) of methodology VII is the same as step (a) of methodology V.

In step (b) of methodology VII, the possible effect of an NCSprotein-targeting drug on animals is evaluated on the basis of theexpression level of the NCS protein gene. Specifically, first, themeasured expression level of the NCS protein gene is checked againstdata on the correlation between the expression level of the NCS proteingene and susceptibility to the NCS protein-targeting drug. Thecorrelation between the expression level of the NCS protein gene andsusceptibility to the NCS protein-targeting drug can be determined by amethod known per se.

Next, from the result of the comparison, susceptibility to the NCSprotein-targeting drug is estimated. It is considered that in animalsexpressing the NCS protein gene at high levels, their susceptibility tothe drug is high (or low), and that in animals expressing the gene atlow levels, their susceptibility is low (or high). Hence, it is possibleto determine the susceptibility of an animal to the NCSprotein-targeting drug by analyzing the expression level of the NCSprotein gene. For example, the likelihood or unlikelihood of obtainmentof desired effect of the drug, or the probability of onset of adverseeffect of a drug, can be determined.

Determination method IV enables a determination of susceptibility to NCSprotein-targeting drug. Hence, determination method IV is useful for,for example, the evaluation of an action of NCS protein-targeting drugon a particular animal, and the like.

8.1.2. Determination Kit for the Onset or Risk of Onset of DiseaseAssociated with NCS Protein-Targeting Drug on the Basis of Measurementof Expression Level of the NCS Protein Gene (Determination Kit IV)

The present invention provides a determination kit that enables the easyconduct of determination method IV.

This determination kit is optionally abbreviated as “determination kitIV.”

In one embodiment, determination kit IV comprises the following (i) and(ii):

-   (i) a means capable of measuring the expression level of the NCS    protein gene;-   (ii) a medium recording the relationship between the effect of NCS    protein-targeting drug and the expression level of the NCS protein    gene.

The constituents of determination kit IV are the same as those ofdetermination kit I except medium (ii).

Determination kit IV enables the easy determination of susceptibility toNCS protein-targeting drug. Hence, determination method IV is usefulfor, for example, the evaluation of an action of NCS protein-targetingdrug on a particular animal, and the like.

8.2. Determination Method and Determination Kit for Susceptibility toNCS Protein-Targeting Drug on the Basis of Identification ofPolymorphism of the NCS Protein Gene

8.2.1. Determination Method for Susceptibility to NCS Protein-TargetingDrug in Disease Associated with NCS Protein-Targeting Drug on the Basisof Identification of Polymorphism of the NCS Protein Gene (DeterminationMethod V)

The present invention provides a determination method for susceptibilityto NCS protein-targeting drug in a disease associated with NCSprotein-targeting drug, which comprises identifying the polymorphism ofthe NCS protein gene.

This determination method is optionally abbreviated as “determinationmethod V.”

In one embodiment, determination method V comprises the following steps(a) and (b):

-   (a) a step for identifying the polymorphism of the NCS protein gene    in a biological sample collected from an animal;-   (b) a step for predicting the effect of NCS protein-targeting drug    in a disease associated with the NCS protein gene on the basis of    the presence or absence of a particular type of polymorphism.

The methodology comprising the above-described steps (a) to (b) isoptionally abbreviated as “methodology VIII.”

Step (a) of methodology VIII is the same as step (a) of methodology VI.

In step (b) of methodology VIII, the effect of NCS protein-targetingdrug in a disease associated with NCS protein-targeting drug isevaluated on the basis of the type of polymorphism of the NCS proteingene. Specifically, first, the identified type of polymorphism of theNCS protein gene is checked against data on the correlation between thetype of polymorphism of the NCS protein gene and susceptibility to NCSprotein-targeting drug in a disease associated with NCSprotein-targeting drug. This correlation can be determined by a methodknown per se.

Next, from the result of the comparison, susceptibility to NCSprotein-targeting drug in a disease associated with NCSprotein-targeting drug is estimated. It is known that in animals thatare highly susceptible to a drug, a particular type of polymorphism isoften observed in the NCS protein gene. Hence, it is possible todetermine the susceptibility of an animal to NCS protein-targeting drugby analyzing polymorphism. For example, the likelihood or unlikelihoodof obtainment of desired effect of the drug, or the probability of onsetof adverse reaction of a drug, can be determined.

Determination method V enables the easy determination of susceptibilityto NCS protein-targeting drug in a disease associated with NCSprotein-targeting drug (for example, central nervous disease). Hence,determination method V is useful for, for example, the evaluation of anaction of NCS protein-targeting drug in a disease associated with NCSprotein-targeting drug, and the like.

8.2.2. Determination Kit for Susceptibility to NCS Protein-TargetingDrug in Disease Associated with NCS Protein-Targeting Drug on the Basisof Identification of Polymorphism of the NCS Protein Gene (DeterminationKit V)

The present invention also provides a determination kit that enables theeasy conduct of determination method V.

This determination kit is optionally abbreviated as “determination kitV.”

In one embodiment, determination kit V comprises the following (i) and(ii):

-   (i) a means capable of identifying the polymorphism of the NCS    protein gene;-   (ii) a medium recording the relationship between the effect of NCS    protein-targeting drug and the polymorphism of the NCS protein gene.

The constituents of determination kit V are the same as those ofdetermination kit II except the medium (ii).

Determination kit V enables a determination of susceptibility to NCSprotein-targeting drug in a disease associated with NCSprotein-targeting drug (for example, central nervous disease). Hence,determination kit V is useful for, for example, the evaluation of anaction of NCS protein-targeting drug in a disease associated with NCSprotein-targeting drug, and the like.

8.2.3. Determination Method for Susceptibility to NCS Protein-TargetingDrug in Disease Associated with the NCS Protein Gene on the Basis ofIdentification of Polymorphism of the NCS Protein Gene (DeterminationMethod VI)

The present invention provides a determination method for susceptibilityto NCS protein-targeting drug in a disease associated with the NCSprotein gene, which comprises identifying the polymorphism of the NCSprotein gene.

This determination method is optionally abbreviated as “determinationmethod VI.”

In one embodiment, determination method VI comprises the following steps(a) and (b):

-   (a) a step for determining the type of polymorphism of the NCS    protein gene in a biological sample collected from an animal;-   (b) a step for predicting the effect of NCS protein-targeting drug    in a disease associated with the NCS protein gene on the basis of    the presence or absence of a particular type of polymorphism.

In this determination method, the type of polymorphism used to determinethe susceptibility is one that alters the ability of NCS protein to bindwith NCS protein-targeting drug. The type of polymorphism can bedetermined by a method known per se such as binding assay. Animalshaving a target gene comprising the type of polymorphism thatpotentiates or reduces the binding ability to the drug are thought to behighly (or poorly) susceptible to the drug; animals having a target genecomprising a type of polymorphism that reduces the binding ability areconsidered to be less (or more) susceptible. Hence, the susceptibilityof an animal to NCS protein-targeting drug can be determined byanalyzing such a type of polymorphism.

The methodology comprising steps (a) and (b) above in determinationmethod VI is the same as methodology VIII except for the type ofpolymorphism of the NCS protein gene to be identified.

Determination method VI enables the easy determination of susceptibilityto NCS protein-targeting drug in a disease associated with NCSprotein-targeting drug. Hence, determination method VI is useful for,for example, the evaluation of an action of NCS protein-targeting drugin a disease associated with NCS protein-targeting drug, and the like.

8.2.4. Determination Kit for the Risk of Onset of Disease Associatedwith the NCS Protein Gene on the Basis of Identification of Polymorphismof the NCS Protein Gene (Determination Kit VI)

The present invention also provides a determination kit that enables theeasy conduct of determination method VI.

This determination kit is optionally abbreviated as “determination kitVI.”

In one embodiment, determination kit VI comprises the following (i) and(ii):

-   (i) a means capable of identifying the polymorphism of the NCS    protein gene;-   (ii) a medium recording the relationship between a disease    associated with the NCS protein gene and the polymorphism of the NCS    protein gene.

In determination kit VI, the type of polymorphism used to determine therisk of onset is one that alters the ability of NCS protein to bind withNCS protein-targeting drug. Such a type of polymorphism can bedetermined by a method known per se such as binding assay.

The constituents of determination kit VI are the same as those ofdetermination kit V except for the type of polymorphism of the NCSprotein gene to be identified.

Determination kit VI enables a determination of susceptibility to NCSprotein-targeting drug in a disease associated with NCSprotein-targeting drug. Hence, determination kit VI is useful for, forexample, the evaluation of an action of NCS protein-targeting drug in adisease associated with NCS protein-targeting drug, and the like.

The disclosures in all publications mentioned herein, including patentsand patent application specifications, are incorporated by referenceherein to the extent that all of them have been given expressly.

EXAMPLES

The present invention is hereinafter described in more detail by meansof the following examples, which, however, are not to be construed aslimiting the present invention.

Reference Example 1 Method of Expressing Proteins from Human Full-LengthcDNA Clone

BP-reaction was performed on human full-length cDNA clone and thecloning vector Gateway pDONR201 by the PCR cloning method using theInvitrogen Gateway system to yield an entry clone. LR-reaction wasperformed on this entry clone with the destination vector pDEST17(Gateway System) and LR Clonase at 25° C. for 60 minutes to yield anexpression plasmid. Escherichia coli competent cell BL21star(DE3)pLysSwere transformed with this expression plasmid, a clone incorporating theexpression vector was selected, and a frozen stock was prepared. Thetransformant was inoculated into LB medium and precultured, after whichit was transferred into SB medium and cultured to induce the expressionof IPTG, and the cells were stored frozen.

Reference Example 2 Method of Purifying the Expressed Protein of HumanFull-Length cDNA Clone

A human full-length cDNA clone was expressed as a protein with anN-terminal His tag. This clone was purified using BioRobot 8000 (Qiagen)or ACTA Crystal (Amersham). In the purification with BioRobot 8000, theexpression-induced frozen stock cells in Reference Example 1 was thawedand lysed with lysozyme, after which the cells were affinity-purifiedusing Ni-NTA Superflow 96 BioRobot Kit (Qiagen). In the purificationwith ACTA Crystal, affinity purification using a HisTrap HP column wasfollowed by gel filtration purification using the Gel Filtration ColumnHiLoad 16/60 or a 10/30 Superdex 75 prep grade column. The purifiedfraction was used for interaction analysis after being subjected toSDS-PAGE to verify the estimated molecular weight and purity.

Reference Example 3 Method of Analyzing Human Protein-Drug InteractionsUsing Size Exclusion Chromatography

To analyze the interactions between commonly used drugs and proteinsexpressed from human full-length cDNA clones while keeping both theproteins and the compounds in non-modified, non-immobilized state, sizeexclusion chromatography (SEC) and mass analysis were used incombination (FIGS. 1, 2). The specific procedures are shown below.

Step 1

A solution of a single drug or a multiplicated compound solutioncomprising a mixture of a plurality of drugs (e.g., 8 kinds, 16 kinds,24 kinds) was added to the protein purified in Reference Example 2.

Step 2

The compound-protein mixture prepared in step 1 was subjected tochromatography using an SEC column, the compound and the protein wereseparated by SEC, and the compound that interacted with the boundcompound or protein contained in the protein fraction was analyzed usinga mass analyzer.

The purified protein standard was concentrated by ultrafiltration andsubjected to buffer solution exchange, and finally concentrated toobtain a concentration of 25 μM or higher in an aqueous solution of 10mM ADA (N-(2-acetamido)iminodiacetic acid) buffer (pH 6.5)-300 mMNaCl-100 μM mineral ion cocktail (Ca(OAc)₂, Zn(OAc)₂.2H₂O, Cu(OAc)₂.H₂O,Co(OAc)₂.4H₂O, Mn(OAc)₂.4H₂O, Mg(OAc)₂.4H₂O, FeCl₃.6H₂O). Proteinconcentrations were measured using BCA Protein Assay (PIERCE), inconsideration of the purity calculated by SDS-PAGE.

A solution of a single pharmaceutical compound at a concentration of1.25 mM in DMSO (dimethyl sulfoxide) or a multiplied compound solutionof a plurality (8 or 16 kinds) of compounds in DMSO was prepared, andthese solutions were used for interaction analysis. In reproducibilityconfirmation experiments or dose dependency determination experiments, asolution of various concentrations of a single compound in DMSO(dimethyl sulfoxide) was used.

Mass analysis was performed using LCQ DECA XP (Thermoelectron) orQ-TOFmicro (Micromass), equipped with an ESI probe. The LC pump used wasAgilent 1100 (Yokogawa Analytical Systems), and the autosampler used wasHTC-PAL (CTC Analytics) equipped with a cooling stacker.

In the SEC method using 384-well spin columns, Unifilter 100 (Whatman),packed with 10 μL (dry volume) of Bio-Gel P6 (BIO-RAD) and swollen withmilliQ water, was used as the SEC column. 13.3 μL of a protein-freereference standard or a 25 μM protein standard and 0.7 μL of amultiplied liquid comprising 25 μM of each pharmaceutical compound (5%DMSO aqueous solution) were mixed; 9 μL of this mixture was aliquotedinto the SEC spin columns. The SEC spin column was mounted on anacetonitrile-aliquoted 384-well U-bottom plate and centrifuged; the SECspin column filtrate, which is a protein fraction, was retrieved in 50%acetonitrile. The protein precipitate produced by the acetonitrile wasremoved via centrifugation and filtration for deproteinization; theresulting filtrate was concentrated by centrifugation and re-dissolvedin 10 μL of 50% methanol to obtain a mass analysis sample. The mobilephase supplied to the mass analyzer was 0.1% formic acid/50% methanolsolution in the positive ion mode, and 0.1% ammonia/50% methanolsolution in the negative ion mode; these mobile phases were used at aflow rate of 40 μL/min. 2-μL of mass analysis samples were injectedusing an autosampler at 2-minute intervals; the mass spectral intensityof the compound was measured to obtain the spectral intensity of thepharmaceutical compound contained in the SEC spin column filtrate(protein fraction eluted from SEC). The protein and the compound werejudged to have interacted with each other if the spectral intensity ofthe compound in a mass analysis sample obtained from an SEC samplesupplemented with a protein standard was greater than the spectralintensity of the compound in a mass analysis sample of reference SECstandard not supplemented with the protein. In the experiments forexamining dose dependency, the protein and the compound were judged tohave interacted with each other dose-dependently if the spectralintensity of the pharmaceutical compound contained in the SEC spincolumn filtrate (protein fraction eluted from SEC) increased as thecompound concentration or/and protein concentration of the SEC samplewas increased.

Example 1 Analysis of Interaction Between FLJ39196 Clone-Derived Proteinand Atorvastatin

The FLJ39196 clone-derived protein was expressed and purified accordingto the methods of Reference Examples 1 and 2, and the interactionbetween the protein expressed and purified from FLJ39196 andatorvastatin was analyzed according to the method of Reference Example3. The results are shown in Table 1. The spectrum intensity of thepharmaceutical compound contained in the SEC spin column filtrate(protein fraction eluted from SEC) increased depending on the doses ofboth atorvastatin and the FLJ39196 expressed protein, from which wedetermined that it was a dose-dependent interaction.

TABLE 1 Measured Mass Range: m/z = 559.25-559.5 compound (μM)FLJ39196-atorvastatin 0 12.5 25 62.5 protein (μM) 0 53987 31597 68345604452 5 56604 247204 110996 2703426 10 117020 569000 597628 3448280 25164530 1285142 2719479 7108230

Hence, the FLJ39196 derived protein was found to be a target protein foratorvastatin, which is a kind of statin compound developed as ananti-cholesterol drug, and which is acknowledge for its effects such asan anti-dementia (Alzheimer) drug. Therefore, a new anti-dementia drugcan be screened by making screening candidate substances interact withthe FLJ39196-derived protein. Specifically, a new anti-dementia(Alzheimer) drug can be screened by, for example, constructing a systemwhich detects the interaction between the FLJ39196-derived protein and acandidate substance according to Example 1.

Example 2 Analysis of Interaction Between FLJ39196 Clone-Derived Proteinand Various Compounds

The FLJ39196 clone-derived protein was expressed and purified accordingto the methods of Reference Examples 1 and 2, and the interactionbetween the protein expressed and purified from FLJ39196 and variouscompounds was analyzed according to the method of Reference Example 3.The results are shown in Table 2 to 28. The spectrum intensity of thepharmaceutical compound-contained in the SEC spin column filtrate(protein fraction eluted from SEC) increased depending on the doses ofboth the FLJ39196-expressed protein and the various compounds, fromwhich we determined that there was a dose-dependent interaction.

TABLE 2 Measured Mass Range: m/z = 461.8-462.8 FLJ39196 - compound (μM)pimozide 0 1 10 100 250 protein (μM) 0 18219277 4813337 4354460 55040679203835 23.75 5818424 6161857 27706749 456093959 1174368945 47.5 50803088507799 142633352 1412301047 2935095895

TABLE 3 Measured Mass Range: total of {circle around (1)}m/z =310.7-311.7 and {circle around (2)}m/z(Na ion adduct) = 332.6-333.6compound (μM) FLJ39196 - Bifonazole 0 1 10 100 250 protein (μM) 038593590 32068857 34579053 30705006 32278650 23.75 37989067 2751635430253800 60323012 85169446 47.5 34294270 33844567 38342358 152159135322219706

TABLE 4 Measured Mass Range: m/z = 298.7-299.7 compound (μM) FLJ39196 -Fendiline 0 1 10 100 250 protein (μM) 0 16096640 18341826 21106520134902492 315067466 23.75 15565227 15884361 18323956 118321460 39697910047.5 5343412 24572438 26691498 256481478 565679716

TABLE 5 Measured Mass Range: m/z = 329.7-330.7 FLJ39196 - compound (μM)chloperastine 0 1 10 100 250 protein (μM) 0 5350191 2920008 13165487276180260 803023959 23.75 3537409 2972794 9288224 218420294 77392450647.5 2912771 4085344 18612563 338617522 1411536255

TABLE 6 Measured Mass Range: m/z = 366.7-367.7 compound (μM) FLJ39196 -Bepridil 0 1 10 100 250 protein (μM) 0 11238373 9581826 14048756358168208 1566782176 23.75 7541774 7408112 35655439 771184721 184100248647.5 6335047 7866658 40680909 1156825217 1990085657

TABLE 7 Measured Mass Range: m/z = 473.7-474.7 FLJ39196 - Raloxifenecompound (μM) hydrochloride 0 1 10 100 250 protein (μM) 0 178433663754698 2330594 7909682 10553153 23.75 4991164 3790373 6024709 124641622157539770 47.5 4375846 3014448 17385466 122918120 666932700

TABLE 8 Measured Mass Range: m/z = 423.2-424.2 FLJ39196 - compound (μM)Benzbromarone 0 1 10 100 250 protein 0 327259 231212 300847 378850461737 (μM) 23.75 301936 357666 1258120 10722991 19795682 47.5 353375412022 1942184 29076230 43413619

TABLE 9 Measured Mass Range: m/z = 324.7-325.7 compound (μM) FLJ39196 -prazepam 0 1 10 100 250 protein (μM) 0 57513356 45379226 21988256130413200 139016191 23.75 28065745 38417587 46660512 258681345 51798322447.5 30688137 26479402 67670753 264065408 706383483

TABLE 10 Measured Mass Range: m/z = 318.7-319.7 FLJ39196 - compound (μM)clotiazepam 0 1 10 100 250 protein (μM) 0 8807574 8089543 19826757251469420 633414607 23.75 7459143 8254340 25038949 283452799 75970689347.5 7176640 9022620 60448900 657940514 1089985810

TABLE 11 Measured Mass Range: m/z = 337.7-338.7 compound (μM) FLJ39196 -Suloctidil 0 1 10 100 250 protein (μM) 0 3542413 6080126 3684060 49690679177432 23.75 6558607 4318261 7828150 163940015 357100809 47.5 48361975977944 7209043 507388292 742015343

TABLE 12 Measured Mass Range: m/z = 411.8-412.8 FLJ39196 - compound (μM)Benzethonium 0 1 10 100 250 protein (μM) 0 22495292 7744717 5781630110985750 3418189873 23.75 8694278 7361705 346972055 58646092376839519322 47.5 9038570 18097692 724595729 9268734340 14536894458

TABLE 13 Measured Mass Range: m/z = 428.9-429.9 FLJ39196 - compound (μM)Bicaltamide 0 1 10 100 250 protein (μM) 0 1319316 196020 269681 38894421804944 23.75 278471 264437 914364 18349511 53664833 47.5 191214 3909464168309 24933110 112816687

TABLE 14 Measured Mass Range: m/z = 430.2-431.2 FLJ39196 - compound (μM)Benzthiazide 0 1 10 100 250 protein 0 1189971 271261 505597 13230311475428 (μM) 23.75 315296 361168 1095223 9348303 25333199 47.5 176906249695 1261413 16067473 28434802

TABLE 15 Measured Mass Range: m/z = 298.7-299.7 compound (μM) FLJ39196 -Minaprine 0 1 10 100 250 protein (μM) 0 16639217 15652447 95722839530918568 1025547751 23.75 9882171 10946604 88615018 7270827111272226745 47.5 8025117 10185140 49245292 427303459 1448860391

TABLE 16 Measured Mass Range: m/z = 407.7-408.7 FLJ39196 - compound (μM)Trifluoperazine 0 1 10 100 250 protein (μM) 0 9858178 6645734 7904072204390563 632906281 23.75 6423833 7580781 40058178 293702761 129298765347.5 5642729 7231665 14142338 5846308 2293500744

TABLE 17 Measured Mass Range: m/z = 315.7-316.7 FLJ39196 - compound (μM)Chlorprothixene 0 1 10 100 250 protein (μM) 0 20818137 12908294 1536859230558420 80321853 23.75 20012122 21615977 31548696 104338961 24335967047.5 18192912 25013147 52145413 207034486 433637183

TABLE 18 Measured Mass Range: m/z = 293.7-294.7 FLJ39196 - compound (μM)Pimethixene 0 1 10 100 250 protein (μM) 0 3544903 2913965 486888758800844 224525009 23.75 3278835 2449690 5737072 121577640 26547564947.5 2729497 2762592 9271060 197624997 359356100

TABLE 19 Measured Mass Range: m/z = 434.7-438.7 FLJ39196 - compound (μM)Flupentixol cis-(Z) 0 1 10 100 250 protein (μM) 0 42590489 5784569059201705 138331819 1019443291 23.75 36874292 38801184 66148127 4747175211437323119 47.5 27636011 54783295 82852252 1301876646 1766098462

TABLE 20 Measured Mass Range: m/z = 473.2-474.2 FLJ39196 - compound (μM)clofazimine 0 1 10 100 250 protein (μM) 0 5613437 1592610 33060681781607 1742291 23.75 1595172 2041846 15886830 138678443 380425425 47.52442639 2399851 57730302 312686548 411373516

TABLE 21 Measured Mass Range: m/z = 327.7-328.7 compound (μM) FLJ39196 -Loxapine 0 1 10 100 250 protein (μM) 0 3522176 3432353 21044859169361258 460296582 23.75 3879570 4528499 11931004 251879811 82542286147.5 2827091 4199361 15635610 339486695 830945491

TABLE 22 Measured Mass Range: m/z = 634.9-635.9 FLJ39196 - compound (μM)Rescinnamine 0 1 10 100 250 protein (μM) 0 11639431 9843174 654489612903374 7315419 23.75 12301790 11269941 21895843 215933519 28884447147.5 13358537 17653515 40677751 675671326 1469409087

TABLE 23 Measured Mass Range: m/z = 666.8-667.8 FLJ39196 - compound (μM)Syrosingopine 0 1 10 100 250 protein (μM) 0 52552166 38126829 4720978441362697 38512184 23.75 56051742 63594361 101347281 465474678 59625031147.5 55701703 70443152 105524442 897067633 1499127775

TABLE 24 Measured Mass Range: m/z = 564.1-565.1 FLJ39196 -Dihydroergotoxine compound (μM) mesylate 0 1 10 100 250 protein (μM) 028173635 25150786 29311635 76261189 326128137 23.75 20474057 1968082336914079 177369018 232715490 47.5 14315178 16556794 45033425 221983211530510575

TABLE 25 Measured Mass Range: m/z = 577.9-578.9 FLJ39196 -Dihydroergotoxine compound (μM) mesylate 0 1 10 100 250 protein (μM) 020191674 17036968 25343556 62894601 234871658 23.75 15759781 25890358111382606 421344902 408003600 47.5 17285522 31988946 207450982 9086980631265975868

TABLE 26 Measured Mass Range: m/z = 611.7-612.7 FLJ39196 -Dihydroergotoxine compound (μM) mesylate 0 1 10 100 250 protein (μM) 033357241 18486648 24024699 30737736 58888872 23.75 25102744 35126832106708813 397014202 267260198 47.5 31509157 40595551 194133547 9310957641112309560

TABLE 27 Measured Mass Range: m/z = 611.9-612.9 FLJ39196 - compound (μM)Dihydroergocristine 0 1 10 100 250 protein (μM) 0 34818618 2566352310810305 75466444 137535150 23.75 28551541 68120944 327393156 910395859871970698 47.5 38121433 95970671 593118709 1961163889 2985742521

TABLE 28 Measured Mass Range: m/z = 328.9-329.9 compound (μM) FLJ39196 -stanozolol 0 1 10 100 250 protein (μM) 0 8793284 3108300 2749518 38304914150230 23.75 3069987 3620481 10223886 127989356 194560574 47.5 25056843989532 25533379 619287498 778914733

Hence, the FLJ39196-derived protein was found to be a target protein forthese various compounds. Therefore, a new drug can be screened by makingscreening candidate substances interact with the FLJ39196-derivedprotein. Specifically, a new drug can be screened by, for example,constructing a system which detects the interaction between theFLJ39196-derived protein and a candidate substance according to themethod of Example 1.

Example 3 Analysis of Interaction Between FLJ20589 Clone-Derived Proteinand Various Compounds

The FLJ20589 clone-derived protein was expressed and purified accordingto the methods of Reference Examples 1 and 2, and the interactionbetween the protein expressed and purified from FLJ20589 and variouscompounds was analyzed according to the method of Reference Example 3.The results are shown in Table 29 to 33. The spectrum intensity of thepharmaceutical compound contained in the SEC spin column filtrate(protein fraction eluted from SEC) increased depending on the doses ofboth the FLJ20589-expressed protein and the various compounds, fromwhich we determined that there was a dose-dependent interaction.

TABLE 29 Measured Mass Range: total of {circle around (1)} m/z =310.8-311.8 and {circle around (2)} m/z(Na ion adduct) = 332.9-333.9compound (μM) FLJ20589 - Bifonazole 0 1 10 100 250 protein (μM) 03600159 2704636 2935358 2558136 3779926 23.75 2324791 2343078 21894893826699 3607174 47.5 2094320 1367901 2513645 8024232 28949362

TABLE 30 Measured Mass Range: m/z = 325.1-326.1 FLJ20589 - compound (μM)Prazepam 0 1 10 100 250 protein 0 696402 563508 752185 5911478 8158416(μM) 23.75 438127 372254 1912944 27662748 53037838 47.5 172968 6030848279573 67316791 87274751

TABLE 31 Measured Mass Range: m/z = 412.0-413.0 FLJ20589 - compound (μM)Benzethonium 0 1 10 100 250 protein (μM) 0 2920130 681042 536408 10191831894576 23.75 852430 567621 13777320 732075554 363052655 47.5 169123179741 1125025 368271326 1844957985

TABLE 32 Measured Mass Range: m/z = 408.1-409.1 FLJ20589 - compound (μM)Trifluoperazine 0 1 10 100 250 protein (μM) 0 1872134 1499445 14208532266965 4146130 23.75 548622 469062 893634 41947038 57062370 47.5 226024459554 2953415 67433540 116228047

TABLE 33 Measured Mass Range: m/z = 405.1-406.1 FLJ20589 - compound (μM)Flunarizine 0 1 10 100 250 protein 0 418270 188851 200579 198757 253795(μM) 23.75 185731 501950 137693 181386 308575 47.5 154411 58869 1781282683794 9317332

Hence, the FLJ20589-derived protein was found to be a target protein forthese various compounds. Therefore, a new drug can be screened by makingscreening candidate substances interact with the FLJ20589-derivedprotein. Specifically, a new drug can be screened by, for example,constructing a system which detects the interaction between theFLJ20589-derived protein and a candidate substance according to themethod of Example 1.

Example 4 Docking Study of FLJ39196 or FLJ20589 Clone-Derived Proteinand Various Compounds

Next, using FLJ39196 or FLJ20589 clone-derived protein as the targetprotein, docking study was performed with the various compoundsmentioned above.

As a result, since the compounds as set forth in formula (I) to (VIII),(1) to (11), and (1′) to (11′) of the present invention or its salts orthe like can bind with FLJ39196 or FLJ20589, it was considered that theycan regulate the function of FLJ39196 or FLJ20589 clone-derived protein.

From the above, the compounds as set forth in formula (I) to (VIII), (1)to (11), and (1′) to (11′) of the present invention or its salts or thelike are considered to be useful for prevention and treatment of subjectdiseases such as dementia and the like, or other purposes mentionedwithin this specification.

Industrial Applicability

The target proteins and target genes of the present invention enable thedevelopment of drugs such as anti-central nervous disease drugs, and thelike. The screening methods of the present invention and the derivativeproduction method of the present invention enable the development ofprophylactic or therapeutic agents for diseases such as central nervousdisease, and investigational reagents for the diseases, and the like.The regulator and derivatives of the present invention can be used forthe prophylaxis and treatment of diseases such as central nervousdisease, and the development of investigational reagents for thediseases, and the like. The complexes and kits of the present inventioncan be used for the screening methods of the present invention, and thelike. The determination methods and determination kits of the presentinvention enable the evaluation of the onset or likelihood of onset ofdiseases in animals, the evaluation of the susceptibility of animals todrugs, and the like.

This application is based on a patent application No. 2004-304864 filedin Japan (filing date: Oct. 19, 2004), the contents of which areincorporated in full herein by this reference.

1. A method for screening a drug, which comprises evaluating whether ornot a test substance regulates the expression or function of an NCSprotein gene by performing the following steps (a) to (c): (a) a stepfor bringing the test substance into contact with the NCS protein ormutant protein thereof; (b) a step for measuring the functional level ofthe NCS protein or mutant protein thereof in the presence of the testsubstance, and comparing said functional level with the functional levelof the NCS protein or mutant protein thereof in the absence of the testsubstance; (c) a step for selecting a test substance that alters thefunctional level of the NCS protein or mutant protein thereof on thebasis of the result of the comparison in (b) above.
 2. The methodaccording to claim 1, wherein the drug regulates central nervous action,dementia action, or Alzheimer's disease action.
 3. The method accordingto claim 1, wherein the drug regulates an action associated with an NCSprotein-targeting drug.
 4. The method according to claim 1, wherein theNCS protein gene is a neurocalcin gene.
 5. The method according to claim1, wherein the NCS protein gene is a neurocalcin δ gene.
 6. The methodaccording to claim 1, wherein the NCS protein or mutant protein thereofis an NCS protein.
 7. A method for screening a drug, which comprisesevaluating whether or not a test substance regulates the expression orfunction of an NCS protein gene by performing the following steps (a) to(c): (a) a step for bringing the test substance into contact with cellsenabling a measurement of the expression of the NCS protein or a geneencoding the NCS protein; (b) a step for measuring the expression levelof the NCS protein or the gene in the cells in contact with the testsubstance, and comparing said expression level with the expression levelof the NCS protein or the gene in control cells not in contact with thetest substance; (c) a step for selecting a test substance that regulatesthe expression level of the NCS protein or the gene on the basis of theresult of the comparison in step (b) above.
 8. The method according toclaim 7, wherein the drug regulates central nervous action, dementiaaction, or Alzheimer's disease action.
 9. The method according to claim7, wherein the drug regulates an action associated with an NCSprotein-targeting drug.
 10. The method according to claim 7, wherein theNCS protein gene is a neurocalcin gene.
 11. The method according toclaim 7, wherein the NCS protein gene is a neurocalcin δ gene.
 12. Amethod for screening a drug, which comprises evaluating whether or not atest substance regulates the expression or function of an NCS proteingene by performing the following steps (a) to (c): (a) a step forbringing the test substance into contact with an NCS protein or a mutantprotein thereof; (b) a step for measuring the ability of the testsubstance to bind to the NCS protein or mutant protein thereof; (c) astep for selecting a test substance that has the binding ability to theNCS protein or mutant protein thereof on the basis of the result of step(b) above.
 13. The method according to claim 12, wherein the drugregulates central nervous action, dementia action, or Alzheimer'sdisease action.
 14. The method according to claim 12, wherein the drugregulates an action associated with an NCS protein-targeting drug. 15.The method according to claim 12, wherein the NCS protein gene is aneurocalcin gene.
 16. The method according to claim 12, wherein the NCSprotein gene is a neurocalcin δ gene.
 17. The method according to claim12, wherein the NCS protein or mutant protein thereof is an NCS protein.18. A method for screening a drug, which comprises evaluating whether ornot a test substance regulates the expression or function of an NCSprotein gene by performing the following steps (a) to (c): (a) a stepfor bringing the test substance and an NCS protein-binding substanceinto contact with an NCS protein or a mutant protein thereof; (b) a stepfor measuring the binding amount of the NCS protein-binding substance tothe NCS protein or mutant protein thereof in the presence of the testsubstance, and comparing the amount with the binding amount of the NCSprotein-binding substance to the NCS protein or mutant protein thereofin the absence of the test substance; (c) a step for selecting a testsubstance that alters the binding amount of the NCS protein-bindingsubstance to the NCS protein or mutant protein thereof on the basis ofthe result of the comparison in step (b) above.
 19. The method accordingto claim 18, wherein the NCS protein-binding substance is atorvastatin,pimozide, bifonazole, flunarizine, fendiline, chloperastine, bepridil,raloxifene hydrochloride, benzbromarone, prazepam, clotiazepam,suloctidil, benzethonium, bicaltamide, benzthiazide, minaprine,trifluoperazine, chlorprothixene, pimethixene, flupentixol, clofazimine,loxapine, rescinnamine, syrosingopine, dihydroergocornine mesylate,dihydro-α-ergocryptine mesylate, dihydro-β-ergocryptine mesylate,dihydroergocristine mesylate or stanozolol, or a derivative thereof. 20.The method according to claim 18, wherein the drug regulates centralnervous action, dementia action, or Alzheimer's disease action.
 21. Themethod according to claim 18, wherein the drug regulates an actionassociated with an NCS protein-targeting drug.
 22. The method accordingto claim 18, wherein the NCS protein gene is a neurocalcin gene.
 23. Themethod according to claim 18, wherein the NCS protein gene is aneurocalcin δ gene.
 24. The method according to claim 18, wherein theNCS protein or mutant protein thereof is an NCS protein.
 25. A methodfor screening for a substance that regulates a function associated withan NCS protein gene, which comprises determining whether or not a testsubstance regulates the binding of an NCS protein-targeting drug to theNCS protein or a mutant protein thereof by performing the followingsteps (a) to (c): (a) a step for bringing the test substance and the NCSprotein-targeting drug into contact with the NCS protein or the mutantprotein thereof; (b) a step for measuring the binding amount of the NCSprotein-targeting drug to the NCS protein or mutant protein thereof inthe presence of the test substance, and comparing the binding amountwith the binding amount of the NCS target drug to the NCS protein ormutant protein thereof in the absence of the test substance; (c) a stepfor selecting a test substance that alters the binding amount of the NCSprotein-targeting drug to the NCS protein or mutant protein thereof onthe basis of the result of the comparison in step (b) above.
 26. Themethod according to claim 25, wherein the NCS protein-targeting drug isatorvastatin, pimozide, bifonazole, flunarizine, fendiline,chioperastine, bepridil, raloxifene hydrochloride, benzbromarone,prazepam, clotiazepam, suloctidil, benzethonium, bicaltamide,benzthiazide, minaprine, trifluoperazine, chlorprothixene, pimethixene,flupentixol, clofazimine, loxapine, rescinnamine, syrosingopine,dihydroergocornine mesylate, dihydro-α-ergocryptine mesylate,dihydro-β-ergocryptine mesylate, dihydroergocristine mesylate orstanozolol, or a derivative thereof having the ability to bind to NCS.27. The method according to claim 25, wherein the NCS protein or mutantprotein thereof is an NCS protein.